Methods and Compositions for Promoting Glucose Homeostasis

ABSTRACT

A method of reducing blood glucose in a subject has been developed. In preferred embodiments, the method involves administering to the subject a specific activator of endogenous mitogen-activated protein kinase kinase 6 (MKK3), mitogen-activated protein kinase kinase 6 (MKK4), mitogen-activated protein kinase kinase 6 (MKK6), p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated kinase-activated protein kinase 2 (MK2), or a combination thereof, in an effective amount to reduce blood glucose in a subject. In other embodiments, the method involves administering to the subject a specific activator to increase X-box binding protein 1 (XBP1) phosphorylation on Thr48 and Ser61 in an effective amount to reduce blood glucose in the subject. Methods of identifying agents for reducing blood glucose in a subject are also provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under AgreementR01DK081009 awarded to Umut Ozcan by the National Institutes of Health.The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is generally related to the field of metabolic homeostasisand insulin resistance, more particularly to methods and compositionsfor reducing endoplasmic reticulum stress, lowering blood glucoselevels, and treating type II diabetes and obesity.

BACKGROUND OF THE INVENTION

For individuals born in the United States in the year 2000, theestimated lifetime risk for developing diabetes is 32.8% for males and38.5% for females, with Hispanic Americans having estimated life timerisks for diabetes of approximately 50% (Narayan, K. M., et al. Jama(2003) 290:1884-1890). Diabetes is the leading cause of adult blindnessand accounts for over 40% of the new cases of end-stage renal disease.The risk of heart disease and stroke is two to four times higher, andthe risk of lower extremity amputation is approximately 20 times higher,for people with diabetes than for those without the disease (Bjork, S.(2001) Diabetes Res Clin Pract 54 Suppl 1:S13-18). Despite its enormousburden on human health and on the global economy, few significantadvances have been made over the last three decades in the therapeutictreatment of type 2 diabetes. Moreover, despite intense researchefforts, the link between obesity and type 2 diabetes is not wellunderstood.

There is an urgent need for effective treatments to reduce ER stressand/or lower blood glucose levels in obese and/or type II diabeticindividuals.

Therefore, it is an object of the invention to provide compositions andmethods for reducing ER stress in obese and/or type II diabeticindividuals.

It is a further object of the invention to provide compositions andmethods for lowering blood glucose levels in obese and/or type IIdiabetic individuals.

SUMMARY OF THE INVENTION

Compositions and methods for reducing blood glucose in a subject havebeen developed. These methods involve increasing p38 MAPK activity inthe liver of obese and diabetic mice in order to significantly increaseX-box binding protein 1 (XBP1) phosphorylation and nucleartranslocation, which markedly enhances glucose tolerance. Prior to thisobservation, the general view in the obesity and diabetes field was thatstress-activated protein kinase (SAPK) signaling is detrimental tometabolic homeostasis.

In some embodiments, the method involves administering to the subject aspecific activator of endogenous mitogen-activated protein kinase kinase3 (MKK3), mitogen-activated protein kinase kinase 4 (MKK4),mitogen-activated protein kinase kinase 6 (MKK6), p38 mitogen-activatedprotein kinase (p38MAPK), mitogen-activated kinase-activated proteinkinase 2 (MK2), or a combination thereof, in an effective amount toreduce blood glucose in the subject. In other embodiments, the methodinvolves administering to the subject a specific activator of X-boxbinding protein 1 (XBP1) in an effective amount to reduce blood glucosein the subject, wherein the specific activator increases phosphorylationof XBP1 on Thr48 and Ser61.

Obesity can lead to endoplasmic reticulum (ER) stress, which in turncontributes to the development of insulin resistance and type-2 diabetesand increased blood glucose levels. Therefore, in preferred embodiments,the subject is obese. In some embodiments, the subject has type-2diabetes or pre-diabetes.

MKK3 is generally activated by phosphorylation at residue Ser189.Therefore, in some embodiments the activator of MKK3 promotesphosphorylation of MKK3 at residue Ser189. MKK4 is generally activatedby phosphorylation at residues Ser257 and Thr261. Therefore, in someembodiments the activator of MKK4 promotes phosphorylation of MKK4 atresidues Ser257, Thr261, or a combination thereof. MKK6 is generallyactivated by phosphorylation at residues Ser207 and Thr211. Therefore,in some embodiments the activator of MKK6 promotes phosphorylation ofMKK6 at residues Ser207, Thr211, or a combination thereof. p38MAPK isgenerally activated by phosphorylation at residues Thr180 and Tyr182.Therefore, in some embodiments the activator of p38MAPK promotesphosphorylation of p38MAPK at residues Thr180, Tyr182, or a combinationthereof. MK2 is generally activated by phosphorylation at residue T334.Therefore, in some embodiments the activator of MK2 promotesphosphorylation of MK2 at residues T334. In other embodiments, thespecific activator of MKK6, p38MAPK, MK2, or a combination thereof, isan allosteric activator.

The pharmaceutical composition is preferably administered at a non-toxicdosage, wherein an effective amount is maintained in the subject's bloodfor a period of at least 5, 6, 7, 8, 9, 10, or more days. For example,in some embodiments, the pharmaceutical composition is an extendedrelease formulation.

Screening systems and methods for identifying an agent that reducesblood glucose in a subject are also provided. In some embodiments, themethod involves contacting a sample containing X-box binding protein 1(XBP1) with a candidate agent and detecting phosphorylation of XBP1,wherein an increase in XBP1 phosphorylation (e.g., at residues Thr48,Ser61, or a combination thereof) compared to a control identifies acandidate agent for reducing blood glucose in a subject. In otherembodiments, the method involves contacting a sample containing XBP1with a candidate agent and detecting cellular localization of XBP1,wherein an increase in XBP1 nuclear translocation compared to a controlidentifies an agent for reducing blood glucose in a subject. In stillother embodiments, the method involves contacting a sample containingp38MAPK with a candidate agent and detecting phosphorylation of p38MAPK,wherein an increase in p38MAPK phosphorylation (e.g., Thr180, Tyr182, ora combination thereof) compared to a control identifies an agent forreducing blood glucose. In still other embodiments, the method involvescontacting a sample containing MK2 with a candidate agent and detectingphosphorylation of MK2, wherein an increase in MK2 phosphorylation(e.g., TT334) compared to a control identifies an agent for reducingblood glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating targets of activation that can promoteXBP1 phosphorylation and nuclear translocation, which markedly enhancesglucose tolerance.

FIG. 2A is a graph showing XBP1s protein/actin ratio (%) in mouseembryonic fibroblasts (MEFs) treated with vehicle (♦) or anisomycin(ANS, ▴) before and at indicated times after addition of cycloheximide(CHX) (10 μg/ml). FIG. 2B is a bar graphs showing XBP1s mRNA levels(normalized to 18S) in MEFs infected with adenoviruses expressing XBP1s(Ad-XBP1s) and subsequently treated with 0, 10, 25, and 50 ng/ml ANS forone hour. FIG. 2C is a graph showing XBP1s mRNA levels (% change) inMEFs infected with Ad-XBP1s and stimulated with ANS (25 ng/ml) for onehour and then treated with actinomycin D (10 μg/ml) for 0, 10, 20, 30,40, 50, and 60 minutes. FIGS. 2D and 2E are bar graphs showing nuclear(FIG. 2D) and cytoplasmic (FIG. 2E) XBP1s protein (ratio of ANS-treatedversus vehicle-treated cells) in MEFs infected with Ad-XBP1s followingexposure to ANS (25 ng/ml) for two hours. Error bars are ±S.E.M.**p<0.01, ***p<0.001.

FIG. 3A is a graph showing XBP1s mRNA levels (% change) in MEFs infectedwith Ad-XBP1s (♦▴), pretreated with SB203580 (10 μM) for 30 min (),stimulated with ANS (25 ng/ml) for an additional one hour (▴) and thentreated with actinomycin D (10 μg/ml) for 0, 10, 20, 30, 40, 50, and 60minutes. FIG. 3B is a bar graph showing XBP1s mRNA levels (normalized to18S) in WT (solid bars) and MK2^(−/−) (shaded bars) MEFs infected withAd-XBP1s and treated with ANS at 0, 10, 25, and 50 ng/ml for one hour.FIG. 3C is a graphs showing XBP1s mRNA levels (% change) inAd-XBP1s-infected WT (♦▴) and MK2^(−/−) (□) MEFs stimulated either withvehicle (♦) or ANS (25 ng/ml) (▴□) and then treated with actinomycin D(10 μg/ml) for 0, 10, 20, 30, 40, 50, and 60 minutes. Error bars are±S.E.M. *p<0.05, ***p<0.001.

FIG. 4 is a tandem mass spectrometry (MS/MS) graph showingphosphorylation of XBP1s at Ser61 after anisomycin stimulation. Thegraph plots relative abundance of XBP1 fragments as a function ofmass-to-charge ratios (m/z).

FIGS. 5A and 5B are bar graphs showing XBP1s (FIG. 5A) and GRP78 (FIG.5B) mRNA levels at fasting and re-fed conditions in the livers of themice either treated with vehicle (solid bars) or p38 MAPK inhibitor(SB203580) (shaded bars). Error bars are ±S.E.M. **p<0.01, ***p<0.001,N/S=Non-significant.

FIGS. 6A and 6B are bar graphs showing blood glucose (mg/dl) (FIG. 6A)and circulating insulin (ng/ml) (FIG. 6B) levels in eight-week-old maleob/ob mice infected with Ad-LacZ (solid bars) or Ad-MKK6Glu (shadedbars) (8×10⁶ pfu/g) via tail vein injection after six hours of fasting.FIG. 6C is a graph showing blood glucose (mg/dl) levels ineight-week-old male ob/ob mice 0, 15, 30, 60, 90, or 120 minutes afterintraperitoneal injection with glucose (0.5 g/kg) five days afterinjection with Ad-LacZ (♦) or Ad-MKK6Glu (▴). FIG. 6D is a bar graphshowing area under the curve (AUC) (min-mg/dl) of data in FIG. 6C formice injected with Ad-LacZ (solid bar) or Ad-MKK6Glu (shaded bar). FIG.6E is a graph showing insulin levels (% change) in eight-week-old maleob/ob mice 0, 15, 30, 60, 90, or 120 minutes after intraperitonealinjection with insulin (2 IU/kg) three days after injection with Ad-LacZ(♦) or Ad-MKK6Glu (▴). FIGS. 6F to 6H are bar graphs showing mRNA levels(normalized to 18s) of XBP1s (FIG. 6F), GRP78 (FIG. 6G) and Erdj4 (FIG.6H) in the livers of Ad-LacZ-injected (solid bars) orAd-MKK6Glu-injected (shaded bars) ob/ob mice. FIGS. 6I to 6K are bargraphs showing the ratio of phosphorylated IR (FIG. 6I), IRS1 (FIG. 6J),and Akt^(Thr308) (FIG. 6K) to total protein in Ad-LacZ-injected (solidbars) or Ad-MKK6Glu-injected (shaded bars) ob/ob mice starved for sixhours on post injection day seven and subsequently infused with insulin(0.75 IU/kg) through portal vein. FIG. 6L is a bar graph showing mRNAlevels (arbitrary units) of GK (first and second bars), PGC1α (third andfourth bars), G6Pase (fifth and sixth bars), and PEPCK (seventh andeighth bars) in the livers of ob/ob mice injected with Ad-LacZ (solidbars) or Ad-MKK6Glu (shaded bars). Error bars are ±S.E.M. *p<0.05,**p<0.01, ***p<0.001.

FIG. 7A is a bar graph showing fasting blood glucose levels (mg/dl) 13days post-injection in XBP1^(flox/flox) mice fed a high fat diet (HFD)for 16 weeks and injected with 1) Ad-LacZ (1.58×10⁸ pfu/g) (solid bar),2) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-LacZ (1.5×10⁸ pfu/g) (shaded bar),or 3) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-Cre (1.5×10⁸ pfu/g) (open bar)though the tail vein. FIG. 7B is a graph showing blood glucose levels(mg/dl) in XBP1^(flox/flox) mice at 0, 15, 30, 60, 90, 120 min afterintraperitoneal glucose (1 g/kg) injection with 1) Ad-LacZ (1.58×10⁸pfu/g) (♦), 2) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-LacZ (1.5×10⁸ pfu/g)(▴), or 3) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-Cre (1.5×10⁸ pfu/g) ().FIG. 7C is a bar graph showing area under the curve (AUC) (min-mg/dl) ofdata in FIG. 7B for mice injected with 1) Ad-LacZ (1.58×10⁸ pfu/g)(solid bar), 2) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-LacZ (1.5×10⁸ pfu/g)(shaded bar), or 3) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-Cre (1.5×10⁸pfu/g) (open bar). FIG. 7D is a bar graph showing the ratio ofp-IRE1^(Ser724) to total IRE1 levels in mice injected with 1) Ad-LacZ(1.58×10⁸ pfu/g) (solid bar), 2) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-LacZ(1.5×10⁸ pfu/g) (shaded bar), or 3) Ad-MKK6Glu (0.08×10⁸ pfu/g) andAd-Cre (1.5×10⁸ pfu/g) (open bar). FIGS. 7E and 7F are bar graphsshowing mRNA levels (arbitrary units) of GRP78 (FIG. 7E, bars 1-3),Erdj4 (FIG. 7E, bars 4-6), GK (FIG. 7F, bars 1-3), G6Pase (FIG. 7F, bars4-6), PEPCK (FIG. 7F, bars 7-9), and PGC1α (FIG. 7F, bars 10-12) in thelivers of mice injected with 1) Ad-LacZ (1.58×10⁸ pfu/g) (solid bar), 2)Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-LacZ (1.5×10⁸ pfu/g) (shaded bar), or3) Ad-MKK6Glu (0.08×10⁸ pfu/g) and Ad-Cre (1.5×10⁸ pfu/g) (open bar).Error bars are ±S.E.M. *p<0.05, **p<0.01, ***p<0.001.

FIG. 8A is a bar graphs showing six-hour fasting blood glucose levels(mg/dl) three days after ob/ob mice were injected with 1) Ad-LacZ (solidbar), 2) Ad-XBP1s (shaded bar), or 3) Ad-XBP1s-T48A/S61A (open bar) viathe tail vein. FIG. 8B is a graph showing blood glucose levels (mg/dl)0, 15, 30, 60, 90, or 120 minutes after intraperitoneal injection withglucose (0.5 g/kg) and five days after injection with 1) Ad-LacZ (♦), 2)Ad-XBP1s (▴), or 3) Ad-XBP1s-T48A/S61A () via the tail vein. FIG. 8C isa bar graph showing area under the curve (AUC) (min-mg/dl) of data inFIG. 8B for mice injected with 1) Ad-LacZ (solid bar), 2) Ad-XBP1s(shaded bar), or 3) Ad-XBP1s-T48A/S61A (open bar). FIG. 8D is a graphshowing insulin levels (% change) in ob/ob mice 0, 15, 30, 60, 90, or120 minutes after intraperitoneal injection with insulin seven daysafter injection with 1) Ad-LacZ (♦), 2) Ad-XBP1s (▴), or 3)Ad-XBP1s-T48A/S61A (). FIGS. 8E to 8G are bar graphs showing mRNAlevels (normalized to 18S) of XBP1s (FIG. 8E), GRP78 (FIG. 8F) and Erdj4(FIG. 8G) in livers of mice injected with 1) Ad-LacZ (solid bar), 2)Ad-XBP1s (shaded bar), or 3) Ad-XBP1s-T48A/S61A (open bar). Error barsare ±S.E.M. *p<0.05, **p<0.01, ***p<0.001.

FIG. 9A is a graph showing mRNA levels (% change) of XBP1s intristetraprolin (TTP) knockout (TTP^(−/−)) cells infected with Ad-XBP1s(♦▴) and subsequently treated with ANS (25 ng/ml) (▴) for one hourfollowed by incubation with actinomycin D (10 μg/ml) for 0, 10, 20, 30,40, 50, and 60 minutes. FIGS. 9B and 9C are bar graphs showing mRNAlevels (normalized to 18S) of AUF1 (FIG. 9B) and KSRP (FIG. 9C) in cellstreated with control pLKO lentiviral vector (FIG. 9B-9C, first bar),lentiviral AUF1 shRNA (FIG. 9B, bars 2-5), or lentiviral KSRP shRNA(FIG. 9B, bars 2-5). Error bars are ±S.E.M. ***p<0.001.

FIG. 10A is a bar graph showing XBP1s mRNA levels (normalized to 18S) inMEFs starved 16 hours in serum-free medium in the absence (bars 1-4) orpresence (bars 5-8) of p38 MAPK inhibitor SB203580 (10 μM) andreincubated in medium containing 10% FBS for 0 (bars 1 and 5), 15 (bars2 and 6), 30 (bars 3 and 7) and 60 (bars 4 and 8) minutes. FIGS. 10B and10C are bar graphs showing XBP1s mRNA levels (normalized to 18S) inwild-type (WT) (FIGS. 10B and 10C, bars 1-4) and p38α^(−/−) (FIG. 10B,bars 5-8), and MKK3,6^(−/−) (FIG. 10C, bars 5-8) cells starved 16 hoursin serum-free medium and reincubated in medium containing 10% FBS for 0(bars 1 and 5), 15 (bars 2 and 6), 30 (bars 3 and 7) and 60 (bars 4 and8) minutes.

FIGS. 11A to 11C are bar graphs showing the ratio of phosphorylated p38MAPK (P-p38) to total p38 protein in the liver (FIG. 11A), muscle (FIG.11B), and adipose (WAT) (FIG. 11C) tissues from age-matched wild-typemale mice under normal diet (shaded bars) or high fat diet (HFD) (soldbars) for 8 weeks. Error bars are ±S.E.M. *p<0.05, **p<0.01.

FIG. 12A is a bar graph showing mRNA level (normalized to 18S) of MKK6the liver (bars 1-2), adipose tissue (WAT) (bars 3-4), and muscle (bars5-6) of 8-week-old male ob/ob mice injected with 8×10⁶ pfu/g Ad-LacZ(solid bar) or Ad-MKK6Glu (shaded bar) through the tail vein. FIGS. 12Band 12C are bar graphs showing blood glucose levels (mg/dl) (FIG. 12B)and body weight (g) of 8-week-old male ob/ob mice before (solid bars)and 4 days after (shaded bars) injection with Ad-LacZ (bars 1-2) orAd-MKK6Glu (bars 3-4). FIGS. 12D and 12E are bar graphs showingaspartate transaminase (AST) (FIG. 12D) and alanine transaminase (ALT)(FIG. 12E) levels in the blood of mice injected with Ad-LacZ (solid bar)or Ad-MKK6Glu (shaded bar). Error bars are ±S.E.M. **p<0.01, ***p<0.001,N/S non-significant.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “subject” or “patient” refers to any individual who is thetarget of administration. The term includes human and veterinarysubjects and does not denote a particular age or sex.

The term “effective amount” refers to a sufficient amount of an agent toprovide a desired effect. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of disease that is being treated, theparticular agent used, and its mode of administration. An appropriate“effective amount” may be determined empirically by one of ordinaryskill in the art using routine methods.

The term “treatment” refers to the medical management of a subject withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “specific activator” as used herein refers to an agent thatbinds and activates endogenous target proteins but does not bind oraffect the activity of other (non-target) proteins at detectable levels.

The term “allosteric” refers to the ability of an agent to regulate anenzyme's activity by binding at a site (i.e., allosteric site) otherthan the enzyme's active site (i.e., orthosteric site). Effectors thatenhance the enzyme's activity are referred to as allosteric activators.Allosteric activation occurs when the binding of the agent enhances theattraction between the enzyme and its substrate.

The term “kinase” refers to an enzyme that modifies other proteins bychemically adding phosphate groups to them (phosphorylation). The termincludes natural and synthetic proteins and peptidomimetics.

The term “phosphatases” refers to an enzyme that removes a phosphategroup from its substrate by hydrolysing phosphoric acid monoesters intoa phosphate ion and a molecule with a free hydroxyl group. The termincludes natural and synthetic proteins and peptidomimetics.

The term “peptidomimetic” refers to non-natural molecules that mimicpeptide structures. They typically arise either from modification of anexisting peptide, or by synthesizing compounds that mimic peptides, suchas peptoids and β-peptides. Modifications include altered backbones andthe incorporation of nonnatural amino acids. The altered chemicalstructure is designed to advantageously adjust the molecular propertiessuch as, stability or biological activity.

The term “promote” refers to a detectable increase in activity, levels,response, condition, or other biological parameter. This includes, forexample, an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or any amount in between, in the activity, levels,response, or condition as compared to the native or control level.

The term “reduce” means to a detectable decrease in activity, levels,response, condition, or other biological parameter. This includes, forexample, a decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or any amount in between, when compared to the native orcontrol level.

The term “glucose homeostasis” refers to the balance of insulin andglucagon to maintain blood glucose levels within normal levels (i.eabout 64.8 to 104.4 mg/dl). An agent that “promotes glucose hemostasis”preferably reduces blood glucose levels down to, but not below, normallevels.

The term “glycogenolysis” refers to the conversion of glycogen polymersto glucose monomers.

The term “gluconeogenesis” refers to the generation of glucose fromnon-carbohydrate carbon substrates such as lactate, glycerol, andglucogenic amino acids.

The term “insulin sensitivity” refers to the amount of insulin needed tolower blood sugar in a subject.

The term “insulin resistance” refers a physiological condition in asubject where insulin becomes less effective at lowering blood sugars(low insulin sensitivity), which results in an increase in bloodglucose. Insulin resistance in muscle and fat cells reduces glucoseuptake, whereas insulin resistance in liver cells results in reducedglycogen synthesis and storage and a failure to suppress glucoseproduction and release into the blood.

The term “type-2 diabetes,” “non-insulin-dependent diabetes mellitus(NIDDM),” and “adult-onset diabetes” refer to a metabolic disordercharacterized by insulin resistance and high blood glucose (e.g.,fasting plasma glucose ≧7.0 mmol/l (126 mg/dl)).

The term “pre-diabetes” refers to a condition that occurs when aperson's blood glucose levels are higher than normal but not high enoughfor a diagnosis of type 2 diabetes.

The term “endoplasmic reticulum (ER) stress” and “unfolded proteinresponse (UPR)” refer to a cellular stress response to an accumulationof unfolded or misfolded proteins in the lumen of the endoplasmicreticulum. The UPR attempts to restore normal function of the cell byhalting protein translation and activate the signaling pathways thatlead to increasing the production of molecular chaperones involved inprotein folding. However, in conditions of prolonged stress, the goal ofthe UPR changes from being one that promotes cellular survival to onethat commits the cell to a pathway of apoptosis. ER stress has also beenshown to link obesity with insulin resistance and type 2 diabetes.

The term “obese” refers to a medical condition in which excess body fathas accumulated to the extent that it may have an adverse effect onhealth, leading to reduced life expectancy and/or increased healthproblems. A person is generally considered obese if their body massindex (BMI) is greater than 30 kg/m² and overweight (pre-obese) if theirBMI is between 25 and 30 kg/m².

The term “percent (%) sequence identity” is defined as the percentage ofnucleotides or amino acids in a candidate sequence that are identicalwith the nucleotides or amino acids in a reference nucleic acidsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

II. Compositions

The endoplasmic reticulum (ER) is responsible for the synthesis ofsecretory and membrane proteins, which acquire their lowest energythree-dimensional structures within this organelle. In addition toprotein synthesis, the ER also plays a key role in lipid and cholesterolbiosynthesis. Perturbations of ER homeostasis due to viral infections,accumulation of unfolded proteins and reduction in the cellular energylevels, or alterations in the capacity of the ER to cope with increasingdemand for protein synthesis, can create a condition referred to as ERstress, and lead to activation of a group of complex signaling pathways,called the unfolded protein response (UPR) (Marciniak, S. J. & Ron, D.(2006) Physiol Rev 86:1133-1149; Schroder, M. & Kaufman, R. J. (2005)Annu Rev Biochem 74:739-789; Ron, D. & Walter, P. (2007) Nat Rev MolCell Biol 8:519-529; Bernales, S., et al. (2006) Annu Rev Cell Dev Biol22:487-508). Two type-I transmembrane kinases (the PKR-like endoplasmicreticulum kinase (PERK) and the inositol requiring enzyme-1 (IRE 1)),plus a specific type-II transmembrane protein (the activatingtranscription factor-6 (ATF6)), have major roles in initiating UPRsignaling. PERK phosphorylates the eukaryotic translation initiationfactor 2 alpha (eIF2α) at Ser51 and leads to a global attenuation in theinitiation of translation, while proteolytic cleavage of ATF6 and itstranslocation to the nucleus increase the expression of genes that areimportant in protein folding and ER homeostasis (Marciniak, S. J. & Ron,D. (2006) Physiol Rev 86:1133-1149; Schroder, M. & Kaufman, R. J. (2005)Annu Rev Biochem 74:739-789; Ron, D. & Walter, P. (2007) Nat Rev MolCell Biol 8:519-529; Bernales, S., et al. (2006) Annu Rev Cell Dev Biol22:487-508).

Of these three transmembrane proteins, IRE1 is the most conservedevolutionarily. In addition to its kinase activity, IRE1 also hasendoribonuclease activity (Cox, J. S., et al. (1993) Cell 73:1197-1206;Mori, K., et al. (1993) Cell 74:743-756). The endoribonuclease domain ofIRE1 cleaves the mRNA of X-Box Binding Protein-1 (XBP1), which is themammalian homolog of yeast Hac1p (Yoshida, H., et al. (2001) Cell107:881-891; Lee, K., et al. (2002) Genes Dev 16:452-466; Calfon, M., etal. (2002) Nature 415:92-96). XBP1 belongs to the CREB/ATF family oftranscription factors and is a basic region-leucine zipper transcriptionfactor (Clauss, I. M., et al. (1996) Nucleic Acids Res 24:1855-1864).IRE1 cleaves the full length XBP1 mRNA to initiate removal of a 26 bpintron, converting the 267 amino acid unspliced XBP1 protein (XBP1u) tothe highly active 371 amino acid spliced XBP1 (XBP1s), a transcriptionfactor that functions as a master regulator of ER folding capacity(Marciniak, S. J. & Ron, D. (2006) Physiol Rev 86:1133-1149; Schroder,M. & Kaufman, R. J. (2005) Annu Rev Biochem 74:739-789; Ron, D. &Walter, P. (2007) Nat Rev Mol Cell Biol 8:519-529; Bernales, S., et al.(2006) Annu Rev Cell Dev Biol 22:487-508). Migration of XBP1s to thenucleus leads to upregulation of gene expression of ER chaperones (Lee,A. H., et al. (2003) Mol Cell Biol 23:7448-7459; Sriburi, R., et al.(2004) J Cell Biol 167:35-41) and of the components of ER-associateddegradation (ERAD). XBP1s also plays a key role in ER expansion(Sriburi, R., et al. (2007) J Biol Chem 282(10):7024-34; Fagone, P., etal. (2007) J Biol Chem 282(10):7591-605).

XBP1s is linked to a number of diseases, including insulin resistanceand type 2 diabetes (Ozcan, U., et al. (2008) Mol Cell 29:541-551;Ozcan, U., et al. (2006) Science 313:1137-1140; Ozcan, U., et al. (2004)Science 306:457-461), leptin resistance and obesity (Ozcan, L., et al.(2009) Cell metabolism 9:35-51), inflammation (Richardson, C. E., et al.(2010) Nature 463:1092-1095; Maranon, F., et al. (2010) Natureimmunology 11:411-418), fatty liver disease (Lee, A. H., et al. (2008)Science 320:1492-1496), neurodegeneration (Sado, M., et al. (2009) Brainresearch 1257:16-24), inflammatory bowel disease (IBD) (Kaser, A., etal. (2008) Cell 134:743-756) and cancer (Koong, A. C., et al. (2006)Cancer Biol Ther 5: 756-759). It is also involved in the regulation of avariety of cellular processes (Ron, D. & Walter, P. (2007) Nat Rev MolCell Biol 8:519-529; Bernales, S., et al. (2006) Annu Rev Cell Dev Biol22:487-508).

Obesity leads to the development of ER stress and activates the UPR inthe liver, adipose tissue and brain (Ozcan, U., et al. (2008) Mol Cell29:541-551; Ozcan, U., et al. (2006) Science 313:1137-1140; Ozcan, U.,et al. (2004) Science 306:457-461; Ozcan, L., et al. (2009) Cellmetabolism 9:35-51; Nakatani, Y., et al. (2005) J Biol Chem 280:847-851;Ozawa, K., et al. (2005) Diabetes 54:657-663), in turn contributing tothe development of insulin resistance, type-2 diabetes and leptinresistance. Reversal of ER stress with chemical chaperones increasesboth insulin and leptin sensitivity (Ozcan, U., et al. (2006) Science313:1137-1140, Ozcan, L., et al. (2009) Cell metabolism 9:35-51).

Nuclear translocation of XBP1s is severely reduced under conditions ofobesity, due to loss of interactions between XBP1s and the p85regulatory subunits (Park, S. W., et al. (2010) Nature Med 16:429-437).Obesity creates an XBP1-deficient condition in the liver. If XBP1s isreactivated in the liver by forced ectopic expression in severely obeseand diabetic mice, blood glucose levels are reduced to euglycemia (Zhou,Y., et al. (2011) Nature Med 17(3):356-65). Thus, unraveling thenetworks that regulate XBP1s activity has the strong potential to revealunique approaches for the treatment of obesity, type 2 diabetes, andother XBP1s-associated diseases.

XBP1s also interacts with the Forkhead box O1 (FoxO1) transcriptionfactor and directs it toward proteasome-mediated degradation. XBP-1s,through its interaction with FoxO1, can bypass hepatic insulinresistance independent of its effects on ER folding capacity.

A. Specific Activators

Specific activators of MKK3, MKK4, MKK6, p38 MAPK, and/or MK2 can reduceblood glucose levels in a subject. In preferred embodiments, thespecific activator is a small molecule.

Mitogen-activated protein kinase kinase 3 (MKK3), also known asmitogen-activated protein kinase kinase 3 and MAP kinase kinase 3 (MAPKK3), is an enzyme that in humans is encoded by the MAP2K3 gene, onchromosome 17. This protein phosphorylates and activates p38mitogen-activated protein kinases (p38 MAPK) kinase.

Mitogen-activated protein kinase kinase 6 (MKK4), also known asmitogen-activated protein kinase kinase 4 and MAP kinase kinase 6 (MAPKK4), is an enzyme that in humans is encoded by the MAP2K4 gene, onchromosome 17. This protein phosphorylates and activates p38mitogen-activated protein kinases (p38 MAPK) kinase.

Mitogen-activated protein kinase kinase 6 (MKK6), also known asmitogen-activated protein kinase kinase 6 and MAP kinase kinase 6 (MAPKK6), is an enzyme that in humans is encoded by the MAP2K6 gene, onchromosome 17. This protein phosphorylates and activates p38mitogen-activated protein kinases (p38 MAPK) kinase in response toinflammatory cytokines or environmental stress.

p38 MAPK is a class of mitogen-activated protein kinases that areresponsive to stress stimuli, such as cytokines, ultravioletirradiation, heat shock, and osmotic shock, and are involved in celldifferentiation and apoptosis. Four isoforms of p38 MAPK have beenidentified: p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12), and p38-δ(MAPK13). p38 MAPK phosphorylates mitogen-activated kinase-activatedprotein kinase 2 (MK2) and X-box binding protein 1 (XBP1) in liver cellsof obese mice.

MK2 is an enzyme that in humans is encoded by the MAPKAPK2 gene. Heatshock protein HSP27 is one of the substrates of this kinase in vivo.

Specific activators of XBP1 that increase phosphorylation on Thr48 andSer61 can also reduce blood glucose in the subject. XBP1 is a proteinwhich in humans is encoded by the XBP1 gene. The XBP1 gene is located onchromosome 22. The XBP1 protein is a transcription factor that regulatesthe expression of genes important to the proper functioning of theimmune system and in the cellular stress response.

1. Allosteric Activators

Positive allosteric modulation (also known as allosteric activation)generally occurs when the binding of one ligand enhances the bindingbetween an enzyme and its substrate. Under normal circumstances, it actsby causing a conformational change in the enzyme, which results in achange in the binding affinity of the enzyme for its substrate.

There are a number of advantages in using allosteric modulators aspreferred therapeutic agents over classic orthosteric ligands. Forexample, these modulators have a decreased potential for toxic effects,since modulators with limited co-operativity will have a ceiling levelto their effect, irrespective of the administered dose. In addition,allosteric modulators are not limited to simply turning a receptor on oroff, the way most drugs are. Instead, they act more like a dimmerswitch, offering control over the intensity of activation ordeactivation, while allowing the body to retain its natural control overinitiating receptor activation.

MKK3, MKK4, and MKK6 bind and phosphorylate p38 MAPK. Therefore, in someembodiments, an allosteric activator of MKK3, MKK4, or MKK6 binds MKK3,MKK4, or MKK6 and enhances binding between MKK3, MKK4, or MKK6 kinasedomain and p38 MAPK and/or phosphorylation of p38MAPK (e.g., at residuesThr180 and/or Tyr182). p38 MAPK binds and phosphorylates XBP1.Therefore, in some embodiments, an allosteric activator of p38 MAPKbinds p38 MAPK and enhances binding between p38 MAPK kinase domain andXBP1 and/or phosphorylation of XBP1 (e.g., at residues Thr48 and/orSer61).

Allosteric activators of MKK3, MKK4, MKK6, p38 MAPK, and/or MK2 can beidentified by first screening compound libraries for compounds that bindMKK3, MKK4, MKK6, p38 MAPK, and/or MK2. These compounds can be furtherscreened to verify that they do not bind MKK3, MKK4, MKK6, p38 MAPK,and/or MK2 at the active (kinase) domain. Candidate compounds can thenbe screened in cell-based or cell-free assays containing MKK3, MKK4,MKK6, p38 MAPK, and/or MK2 and the appropriate substrates underconditions suitable for substrate phosphorylation. Substratephosphorylation can be determined directly (e.g., phosphor-specificantibodies) or indirectly (e.g., activity of substrate enzyme).

2. Kinase Activators

MKK3, MKK4, MKK6, p38 MAPK, MK2, and XBP1 are each activated byphosphorylation of amino acid residues by kinases. Therefore, in someembodiments, the specific activator of MKK3, MKK4, MKK6, p38 MAPK,and/or MK2 is a kinase. In some embodiments, the specific activator thatincreases XBP1 phosphorylation is a kinase.

The kinase can be a naturally occurring protein or variant thereof.Protein variants include peptide fragments, peptidomimetics, andrecombinant proteins (including fusion proteins).

For example, MKK3, MKK4, and MKK6 activate p38 MAP kinase byphosphorylation at Thr180 and Tyr182. Therefore, the specific activatorof p38 MAPK can be a dual-specific protein kinase having the EnzymeCommission number (EC number) 2.7.12.2. In some embodiments, thespecific activator of p38 MAPK is MKK3, MKK4, or MKK6, or a fragment orvariant thereof that phosphorylates p38 MAPK. p38 MAP kinase activatesXBP1 by phosphorylation at Thr48 and Ser61. In some embodiments, thespecific activator of XBP1 is p38 MAP kinase, or a fragment or variantthereof that phosphorylates XBP1. Suitable peptide fragments preferablyinclude at least the kinase domain and protein binding domain of thenatural protein.

Kinase activators can also be identified by screening compoundlibraries. In some embodiments, the library is first screened forcompounds that bind MKK3, MKK4, MKK6, p38 MAPK, MK2, or XBP1 at thephosphorylation site(s) and/or displaces a natural kinase from MKK3,MKK4, MKK6, p38 MAPK, MK2, or XBP1. For example, a compound library canbe screened for compounds that bind p38 MAPK at Thr180 and/or Tyr182 orfor compounds that displace MKK3, MKK4, or MKK6 from p38 MAPK.Alternatively, or in addition, compound libraries, or candidate agentsfrom the prior screen, can be screened for compounds that phosphorylateMKK3, MKK4, MKK6, p38 MAPK, MK2, or XBP1. Compound libraries orcandidate agents can also be screened for the ability to activate MKK3,MKK4, MKK6, p38 MAPK, MK2, or XBP1 and induce XBP1 nuclearinternalization in vitro or in vivo.

Allosteric and kinase activators identified for use in the disclosedmethods are preferably also screened against other map kinase substratesto determine if the compound's activity is specific for MKK3, MKK4,MKK6, p38 MAPK, MK2, or XBP1. Compounds that substantially activate orinhibit other enzymes are non-specific and are more likely to causeside-effects and toxicity. Candidate activators are therefore alsopreferably tested in vivo for therapeutic effect, side-effects, andtoxicity.

3. Peptides and Peptidomimetics

Endogenous MKK3, MKK4, MKK6, p38 MAPK, and/or MK2 activity can also besupplemented using exogenous MKK3, MKK4, MKK6, p38 MAPK, and/or MK2protein (peptide), or variants thereof that activate target substrate.The peptide or peptidomimetics of MKK3, MKK4, MKK6, p38 MAPK, and/or MK2can be administered to subjects in a formulation that permits entry ofthe peptides or peptidomimetics into liver cells of the subject.

a. Peptide Variants

Protein variants and derivatives are well understood to those of skillin the art and in can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Insertions ordinarily will be smaller insertions than those ofamino or carboxyl terminal fusions, for example, on the order of one tofour residues.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of sequence identity to specific known sequences.Specifically disclosed are variants of these and other proteins hereindisclosed which have at least 70%, 75%, 80%, 85%, 90% or 95% sequenceidentity to the endogenous sequence. Those of skill in the art readilyunderstand how to determine the sequence identity of two proteins. Forexample, the sequence identity can be calculated after aligning the twosequences so that the sequence identity is at its highest level.

b. Peptidomimetics

Peptidomimetics have general features analogous to their parent peptidestructures, such as enzyme activity. The peptidomimetic materials can beclassified into five categories: α-peptides, β-peptides, γ-peptides,δ-peptides, and oligomers having backbones which can adopt helical orsheet conformations. Copolymers of these peptides can also be used.

Examples of α-peptide peptidomimetics include, but are not limited to,N,N′-linked oligoureas, oligopyrrolinones, oxazolidin-2-ones, azatidesand azapeptides. Examples of β-peptides include, but are not limited to,peptide foldamers, aminoxy acids, sulfur-containing peptide analogues,and hydrazino peptides. Examples of γ-peptides include, but are notlimited to, peptide foldamers, oligoureas, oligocarbamates, andphosphodiesters. Examples of δ-peptides include, but are not limited to,alkene-based amino acids and carbopeptoids, such as pyranose-basedcarbopeptoids and furanose-based carbopeptoids.

Another class of compounds includes oligomers having backbones which canadopt helical or sheet conformations. Example of such compounds include,but are not limited to, compounds having backbones utilizing bipyridinesegments, compounds having backbones utilizing solvophobic interactions,compounds having backbones utilizing side chain interactions, compoundshaving backbones utilizing hydrogen bonding interactions, and compoundshaving backbones utilizing metal coordination.

Examples of compounds containing backbones utilizing bipyridine segmentsinclude, but are not limited to, oligo(pyridine-pyrimidines),oligo(pyridine-pyrimidines) with hydrazal linkers, andpyridine-pyridazines.

Examples of compounds containing backbones utilizing solvophobicinteractions include, but are not limited to, oligoguanidines, aedamers(structures which take advantage of the stacking properties of aromaticelectron donor-acceptor interactions of covalently linked subunits) suchas oligomers containing 1,4,5,8-naphthalene-tetracarboxylic diimiderings and 1,5-dialkoxynaphthalene rings, and cyclophanes such assubstituted N-benzyl phenylpyridinium cyclophanes.

Examples of compounds containing backbones utilizing side chaininteractions include, but are not limited to, oligothiophenes such asoligothiophenes with chiral p-phenyl-oxazoline side chains, andoligo(m-phenylene-ethynylene)s.

Examples of compounds containing backbones utilizing hydrogen bondinginteractions include, but are not limited to, aromatic amide backbonessuch as oligo(acylated 2,2′-bipyridine-3,3′-diamine)s andoligo(2,5-bis[2-aminophenyl]pyrazine)s, diaminopyridine backbonestemplated by cyanurate, and phenylene-pyridine-pyrimidine ethynylenebackbones templated by isophthalic acid.

Examples of compounds containing backbones utilizing metal coordinationinclude, but are not limited to, zinc bilinones, oligopyridinescomplexed with Co(II), Co(III), Cu(II), Ni(II), Pd(II), Cr(III), orY(III), oligo(m-phenylene ethynylene)s containing metal-coordinatingcyano groups, and hexapyrrins.

c. Protein Transduction Domains

The proteins or protein variants can be linked to an internalizationsequence or a protein transduction domain to effectively enter the cell.Recent studies have identified several cell penetrating peptides,including the TAT transactivation domain of the HIV virus, antennapedia,and transportan that can readily transport molecules and small peptidesacross the plasma membrane. Polyarginine has shown an even greaterefficiency of transporting peptides and proteins across the plasma,membrane making it an attractive tool for peptide mediated transport.Non-arginine has been described as one of the most efficientpolyarginine based protein transduction domains, with maximal uptake ofsignificantly greater than TAT or antennapeadia. Peptide mediatedcytotoxicity has also been shown to be less with polyarginine-basedinternalization sequences. R₉ mediated membrane transport is facilitatedthrough heparan sulfate proteoglycan binding and endocytic packaging.Once internalized, heparan is degraded by heparanases, releasing R₉which leaks into the cytoplasm. Studies have recently shown thatderivatives of polyarginine can deliver a full length p53 protein tooral cancer cells, suppressing their growth and metastasis, definingpolyarginine as a potent cell penetrating peptide.

Thus, the provided polypeptide can contain a cellular internalizationtransporter or sequence. The cellular internalization sequence can beany internalization sequence known or newly discovered in the art, orconservative variants thereof. Non-limiting examples of cellularinternalization transporters and sequences include Polyarginine (e.g.,R₉), Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antpmutant), Buforin II, Transportan, MAP (model amphipathic peptide),K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC(Bis-Guanidinium-Spermidine-Cholesterol, and BGTC(Bis-Guanidinium-Tren-Cholesterol)

B. Phosphatase Inhibitors

Another way to increase activity of MKK3, MKK4, MKK6, p38 MAPK, MK2,and/or XBP1 is to inhibit dephosphorylation of the endogenous proteins.Phosphatases are enzymes that remove the phosphate group from substrateproteins that were added by kinases. Therefore, in some embodiments,inhibitors of phosphatases for MKK3, MKK4, MKK6, p38 MAPK, MK2, and/orXBP1 can be used in the disclosed methods to increase MKK3, MKK4, MKK6,p38 MAPK, MK2, and/or XBP1 activity.

For example, protein phosphatase 2A (PP2A) has been shown todephosphorylate p38 MAPK in some cells. Therefore, in some embodiments,a specific inhibitor of PP2A can increase p38 MAPK activity.Phosphatases that dephosphorylate MKK3, MKK4, MKK6, p38 MAPK, MK2,and/or XBP1 in liver cells of obese subjects can be identified usingroutine skill. Moreover, specific inhibitors of many phosphatases havebeen identified and are commercially available.

Phosphatase inhibitors can also be identified by screening compoundlibraries. As above, in some embodiments, the library is first screenedfor compounds that bind MKK3, MKK4, MKK6, p38 MAPK, MK2, and/or XBP1 atthe phosphorylation site(s) and/or displaces a natural kinase orphosphatase from MKK3, MKK4, MKK6, p38 MAPK, MK2, and/or XBP1.Alternatively, or in addition, compound libraries, or candidate agentsfrom the prior screen, can be screened for compounds thatdephosphorylate MKK3, MKK4, MKK6, p38 MAPK, MK2, and/or XBP1. Compoundlibraries or candidate agents can also be screened for the ability toinhibit MKK3, MKK4, MKK6, p38 MAPK, or MK2 activity and inhibit XBP1phosphorylation and/or nuclear internalization in vitro or in vivo.

Phosphatase inhibitors identified for use in the disclosed methods arepreferably also screened against other map kinase substrates todetermine if the compound's activity is specific for phosphatases ofMKK3, MKK4, MKK6, p38 MAPK, MK2, and/or XBP1. Compounds thatsubstantially activate or inhibit other enzymes are non-specific and aremore likely to case side-effects and toxicity. Candidate phosphataseinhibitors are therefore also preferably tested in vivo for therapeuticeffect, side-effects, and toxicity.

C. Pharmaceutical Compositions

Pharmaceutical formulations of the disclosed specific activators ofMKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1, or other disclosedtherapeutic compounds or agents can be used therapeutically incombination with a pharmaceutically acceptable carrier. The disclosedtherapeutic compounds can be incorporated in pharmaceutical formulationsas neutral compounds, pharmaceutically acceptable salts, and/orprodrugs. Pharmaceutical formulations can be designed for immediaterelease, sustained release, delayed release and/or burst release of oneor more specific activators of MKK3, MKK4, MKK6, p38MAPK, MK2, and/orXBP1 in a therapeutically effective amount. In a preferred embodiment,the formulation provides an initial burst release of a “loading dosage”,followed by a sustained release to maintain the therapeuticallyeffective dosage. This can be accomplished using a delayed and/orextended release formulation.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). Pharmaceutical carriers areknown to those skilled in the art. These most typically would bestandard carriers for administration of drugs to humans, includingsolutions such as sterile water, saline, and buffered solutions atphysiological pH.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, and surface active agents.

In some embodiments, the compositions may be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

The pharmaceutical compositions can be formulated for any route ofadministration suitable for targeting liver cells in a subject. Inpreferred embodiments, the pharmaceutical compositions are formulatedfor parental or oral administration.

1. Formulations for Parenteral Administration

The compounds described herein can be formulated for parenteraladministration. Parenteral formulations can be prepared as aqueouscompositions using techniques is known in the art. Typically, suchcompositions are prepared as injectable formulations, for example,solutions or suspensions; solid forms suitable for using to preparesolutions or suspensions upon the addition of a reconstitution mediumprior to injection; emulsions, such as water-in-oil (w/o) emulsions,oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, oremulsomes. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, one or more polyols (e.g., glycerol,propylene glycol, and liquid polyethylene glycol), oils, such asvegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), andcombinations thereof.

Solutions and dispersions of the active compounds as the free acid orbase or pharmacologically acceptable salts thereof can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents, andcombination thereof.

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

The parenteral formulations described herein can be formulated forcontrolled release including immediate release, delayed release,extended release, pulsatile release, and combinations thereof.

For example, the compounds and/or one or more additional active agentscan be incorporated into polymeric microparticles which providecontrolled release of the drug(s). Release of the drug(s) is controlledby diffusion of the drug(s) out of the microparticles and/or degradationof the polymeric particles by hydrolysis and/or enzymatic degradation.Suitable polymers include ethylcellulose and other natural or syntheticcellulose derivatives. Polymers which are slowly soluble and form a gelin an aqueous environment, such as hydroxypropyl methylcellulose orpolyethylene oxide may also be suitable as materials for drug containingmicroparticles. Other polymers include, but are not limited to,polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such aspolylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)(PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof.

Alternatively, the drug(s) can be incorporated into microparticlesprepared from materials which are insoluble in aqueous solution orslowly soluble in aqueous solution, but are capable of degrading withinthe GI tract by means including enzymatic degradation, surfactant actionof bile acids, and/or mechanical erosion. As used herein, the term“slowly soluble in water” refers to materials that are not dissolved inwater within a period of 30 minutes. Preferred examples include fats,fatty substances, waxes, wax-like substances and mixtures thereof.Suitable fats and fatty substances include fatty alcohols (such aslauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids andderivatives, including, but not limited to, fatty acid esters, fattyacid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats.Specific examples include, but are not limited to hydrogenated vegetableoil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenatedoils available under the trade name Sterotex®, stearic acid, cocoabutter, and stearyl alcohol. Suitable waxes and wax-like materialsinclude natural or synthetic waxes, hydrocarbons, and normal waxes.Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material which is normally solid at roomtemperature and has a melting point of from about 30 to 300° C.

2. Oral Formulations

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders, and surfactants, may be desirable.

Diluents, also termed “fillers,” are typically necessary to increase thebulk of a solid dosage form so that a practical size is provided forcompression of tablets or formation of beads and granules. Suitablediluents include, but are not limited to, dicalcium phosphate dihydrate,calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose,microcrystalline cellulose, kaolin, sodium chloride, dry starch,hydrolyzed starches, pre-gelatinized starch, silicone dioxide, titaniumoxide, magnesium aluminum silicate and powder sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pre-gelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone. Some of thematerials which are suitable as binders can also be used asmatrix-forming materials such as hydroxypropyl methyl cellulose, ethylcellulose, and microcrystalline cellulose.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pre-gelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium salts of longchain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads, granules or particles may also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, and preservatives.

Delayed release in an oral formulation can be achieved using entericcoatings. The enteric coated formulation remains intact or substantiallyintact in the stomach but dissolves and releases the contents of thedosage form once it reaches the small intestine. Other types of coatingscan be used to provide delayed release following injectionsubcutaneously, intra-tissue or intramuscularly at a site near or at thearea to be treated.

3. Nasal, Pulmonary or Other Mucosal Formulations

The disclosed compositions can be formulated for delivery to therespiratory system or other mucosa. Suitable formulations foradministration of pharmaceutical compositions to the respiratory systeminclude nasal and pulmonary formulations. For example, compositions canbe delivered to the respiratory system from an inhalation device,including a dry powder inhaler (DPI), metered-dose-inhaler (MDI),nebulizer, or by an instillation technique. Various suitable devices andmethods of inhalation which can be used to administer compositions to apatient's respiratory tract are known in the art, including, but are notlimited to, the Spinhaler® (Fisons, Loughborough, U.K.), Rotahaler®(Glaxo-Wellcome, Research Triangle Technology Park, N.C.), FlowCaps®(Hovione, Loures, Portugal), Inhalator® (Boehringer-Ingelheim, Germany),the Aerolizer® (Novartis, Switzerland), the diskhaler (Glaxo-Wellcome,RTP, NC).

A formulation for intranasal administration may include solutions,suspensions or emulsions of the active compound in a liquid carrier inthe form of drops, mists, sprays, aerosols, or atomizers. Suitableliquid carriers include water, propylene glycol and otherpharmaceutically acceptable alcohols. The formulation may be sterilized,as required. The formulation may also contain adjuvants such aspreservatives, stabilizers, emulsifiers or suspending agents, wettingagents, salts for varying the osmotic pressure or buffers, as required.The nasal formulations may also be administered in the form of a powder.For example, a powdery nasal composition can be directly used as apowder for a unit dosage form. If desired, the powder can be filled incapsules such as hard gelatine capsules. The contents of the capsule orsingle dose device may be administered using e.g. an insufflator.Preferably, it is provided with means ensuring dosing of a substantiallyfixed amount of composition/actuation.

D. Kits

One or more of the compositions described herein can be assembled inkits, together with instructions for use. Kits can include one or morecontainers containing a pharmaceutical composition including atherapeutically effective amount of a specific activator of MKK3, MKK4,MKK6, p38 MAPK, MK2, and/or XBP1. Such kits can further include, ifdesired, one or more of various conventional pharmaceutical kitcomponents, such as, for example, containers with one or morepharmaceutically acceptable carriers as will be readily apparent tothose skilled in the art. The kit may also include means ofadministration, such as one or more of a syringe (e.g., a barrel syringeor a bulb syringe), intravenous (IV) bag, IV line, IV needle, and/orcannula. Printed instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

III. Methods

A. Reducing Blood Glucose

The disclosed pharmaceutical compositions can be used to reduce bloodglucose levels in a subject with high blood sugar. Normal fastingglucose levels are generally in the range of about less than 110 mg/dL.Shortly after eating, the blood glucose level may rise temporarily up to140 mg/dL. Fasting blood glucose levels over 126 mg/dL, and plasmaglucose 2 hours after eating over 200 mg/dL are indicative of metabolicdisorders, such as type-2 diabetes. Therefore, in preferred embodiments,the pharmaceutical compositions are administered in amounts effective toreduce fasting blood glucose levels in the subject to less than 130mg/dL, preferably less than 110 mg/dL, and/or the plasma glucose 2 hoursafter eating to less than 200 mg/dL, preferably less than 140 mg/dL.

Efficacy of the disclosed methods can be monitored by measuring changesin blood glucose levels, body weight, O₂ consumption, and % and totalbody fat content. A statistically significant change in any of theseparameters can be considered evidence of therapeutic efficacy. It ispreferred that a given marker change by at least 5%, at least 10%, atleast 20%, at least 30%, at least 50% or more in effective therapy.Dosage of the pharmaceutical compositions can be modified by thephysician to increase efficacy while avoiding side effects or toxicity.

B. Administration

The disclosed pharmaceutical compositions may be administered in anumber of ways. The compositions are preferably administeredparenterally or orally. In some embodiments, the disclosed compositionsmay be administered transdermally, ophthalmically, vaginally, rectally,pulmonary or intranasally.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Examples of parental administration includeintravenous, intraperitoneal, intramuscular, subcutaneous, andintracavity injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. Parenteral administration can also involve use of a slowrelease or sustained release system such that a constant dosage ismaintained.

The disclosed pharmaceutical compositions may also be administeredprophylactically to subjects. Thus, the disclosed methods can furtherinvolve the step of identifying a subject at risk for a disease ordisorder prior to administration of the disclosed compositions.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the allergic disorder being treated, theparticular compound or agent used, and its mode of administration. Anappropriate amount can be determined by one of ordinary skill in the artusing routine experimentation. For example, effective dosages andschedules for administering the compositions may be determinedempirically, and making such determinations is within the skill in theart. The dosage ranges for the administration of the compositions arethose large enough to produce a desired effect. The dosage should not beso large as to cause adverse side effects, such as liver toxicity.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient, route of administration, or whether otherdrugs are included in the regimen. The dosage can be adjusted by theindividual physician in the event of any counter indications. Unitdosages can vary depending on the frequency of administration. Forexample, the composition can be administered in one or more doseadministrations daily, for one or several days. Guidance can be found inthe literature for appropriate dosages for given classes ofpharmaceutical products.

In one embodiment, specific activators of MKK3, MKK4, MKK6, p38 MAPK,MK2, and/or XBP1 are administered in a dose equivalent to parenteraladministration of about 0.1 ng to about 100 g per kg of body weight,about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1g per kg of body weight, from about 1 μg to about 100 mg per kg of bodyweight, from about 1 μg to about 50 mg per kg of body weight, from about1 mg to about 500 mg per kg of body weight; and from about 1 mg to about50 mg per kg of body weight. Alternatively, the amount of specificactivators of MKK3, MKK4, MKK6, p38 MAPK, MK2, and/or XBP1 administeredto achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng,100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg,19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg,500 mg per kg of body weight or greater.

The specific activators of MKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1may be administered one or more times a day. The duration of thetreatment may be once per day for a period of about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 days or more. Subsequent dosage units can be administered anytime following the initial administration such that a therapeutic effectis achieved.

Area under the curve (AUC) refers to the serum concentration (nmol/L) ofspecific activators of MKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1 overa given time following the IV administration of the reference specificactivators of MKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1 standard. By“reference specific activators” is intended a formulation of specificactivators of MKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1 that serves asthe basis for determination of the total specific activators of MKK3,MKK4, MKK6, p38MAPK, MK2, and/or XBP1 dose to be administered to a humansubject to achieve the desired positive effect, i.e., a positivetherapeutic response that is improved with respect to that observedwithout administration of specific activators of MKK3, MKK4, MKK6,p38MAPK, MK2, and/or XBP1.

In a preferred embodiment, the dose of specific activators of MKK3,MKK4, MKK6, p38MAPK, MK2, and/or XBP1 to be administered provides afinal serum level of specific activators of MKK3, MKK4, MKK6, p38MAPK,MK2, and/or XBP1 of about 100 ng/ml to about 1000 ng/ml, about 1100ng/ml to about 1450 ng/ml, 100 ng/ml to about 250 ng/ml, about 200 ng/mlto about 350 ng/ml, about 300 ng/ml to about 450 ng/ml, about 350 ng/mlto about 450 ng/ml, about 400 ng/ml to about 550 ng/ml, about 500 ng/mlto about 650 ng/ml, about 600 ng/ml to about 750 ng/ml, about 700 ng/mlto about 850 ng/ml, about 800 ng/ml to about 950 ng/ml, about 900 ng/mlto about 1050 ng/ml, about 1000 ng/ml to about 1150 ng/ml, about 100ng/ml to about 1250 ng/ml, about 1200 ng/ml to about 1350 ng/ml, about1300 ng/ml to about 1500 ng/m. In specific embodiments, the serum levelof specific activators of MKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1 isabout 100 ng/ml, 250 ng/ml, 300 ng/ml, 350 ng/ml, 360 ng/ml, 370 ng/ml,380 ng/ml, 390 ng/ml, 400 ng/ml, 410 ng/ml, 420 ng/ml, 430 ng/ml, 440ng/ml, 450 ng/ml, 500 ng/ml, 750 ng/ml, 900 ng/ml, 1200 ng/ml, 1400ng/ml, or 1600 ng/ml.

In some embodiments, the final serum level of specific activators ofMKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1 is at least an amounteffective to detectably increase phosphorylation of MKK3, MKK4, MKK6,p38MAPK, MK2, and/or XBP1 in liver cells of the subject.

C. Screening Methods

Screening systems and methods for identifying an agent that reducesblood glucose in a subject are also provided. In some embodiments, themethod involves contacting MKK3, MKK4, MKK6, p38MAPK, MK2, and/or XBP1proteins (or fragments thereof) with a candidate agent and assaying foran effect, such as binding, phosphorylation, dephosphorylation, andkinase activity. In addition, XBP1 can be assayed for activation, e.g.,nuclear localization. In some embodiments, the method involvescontacting a sample containing X-box binding protein 1 (XBP1) with acandidate agent and assaying for an effect, such as binding,phosphorylation, dephosphorylation, and kinase activity. In someembodiments, the method involves detecting phosphorylation of XBP1,wherein an increase in XBP1 phosphorylation (e.g., at residues Thr48,Ser61, or a combination thereof) compared to a control identifies acandidate agent for reducing blood glucose in a subject. In otherembodiments, the method involves contacting a sample containing XBP1with a candidate agent and detecting cellular localization of XBP1,wherein an increase in XBP1 nuclear translocation compared to a controlidentifies an agent for reducing blood glucose in a subject. In stillother embodiments, the method involves contacting a sample containingp38MAPK with a candidate agent and detecting phosphorylation of p38MAPK,wherein an increase in p38MAPK phosphorylation (e.g., Thr180, Tyr182, ora combination thereof) compared to a control identifies an agent forreducing blood glucose. In still other embodiments, the method involvescontacting a sample containing MK2 with a candidate agent and detectingphosphorylation of MK2, wherein an increase in MK2 phosphorylation(e.g., TT334) compared to a control identifies an agent for reducingblood glucose.

1. Binding

The binding of candidate agents to MKK3, MKK4, MKK6, p38MAPK, MK2, orXBP1 can be detected using routine methods, such as immunodetectionmethods, that do not disturb protein binding. The methods can becell-based or cell-free assays. Immunoassays, in their most simple anddirect sense, are binding assays involving binding between antibodiesand antigen. Many types and formats of immunoassays are known and allare suitable for detecting the disclosed biomarkers. Examples ofimmunoassays are enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA), radioimmune precipitation assays (RIPA),immunobead capture assays, Western blotting, dot blotting, gel-shiftassays, Flow cytometry, protein arrays, multiplexed bead arrays,magnetic capture, in vivo imaging, fluorescence resonance energytransfer (FRET), and fluorescence recovery/localization afterphotobleaching (FRAP/FLAP).

2. Phosphorylation

A classical method of directly detecting protein phosphorylationinvolves the incubation of whole cells with radiolabeled³²P-orthophosphate, the generation of cellular extracts, separation ofproteins by SDS-PAGE, and exposure to film. Other traditional methodsinclude 2-dimensional gel electrophoresis, a technique that assumesphosphorylation will alter the mobility and isoelectric point of theprotein. More recently, the development of phosphorylationstate-specific (phospho-specific) antibodies have provided a means todetect phosphorylation of specific amino acid residues. The antibodiesare generally produced by immunizing animals with syntheticphosphopeptides representing the amino acid sequence surrounding thephosphorylation site of the target protein. The immune sera is appliedto a peptide affinity column to generate a highly specificimmunoreagent. These phospho-specific antibodies are then used inimmunoassays to detect phosphorylated protein.

3. Kinase Activity

Protein kinases are often common elements in multiple signaling networksinfluencing numerous downstream effectors responsible for a biologicalresponse. Kinase activity within a biological sample is commonlymeasured in vitro by incubating the immunoprecipitated kinase with anexogenous substrate in the presence of ATP. Measurement of thephosphorylated substrate can be assessed by several reporter systemsincluding colorimetric, radioactive, or fluorometric detection. Althoughinformation can be obtained regarding the actions of a specific kinase,assessing enzyme activity in cellular extracts only provides a glimpseof the signaling landscape. Little is revealed about the proteins beingmodified, and in vitro activity assays do not address the role ofpotential endogenous phosphatase activity. Direct detection ofphosphorylated proteins can provide a more detailed analysis of thecellular response to an external stimulus, as identification of aphosphopeptide provides information regarding the expression and thefunctional state of that protein.

4. Candidate Agents

In general, candidate agents can be identified from large libraries ofnatural products or synthetic (or semi-synthetic) extracts or chemicallibraries according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s). Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited to,plant-based, fungal-based, prokaryotic-based, or animal-based extracts,fermentation broths, and synthetic compounds, as well as modification ofexisting compounds. Numerous methods are also available for generatingrandom or directed synthesis (e.g., semi-synthesis or total synthesis)of any number of chemical compounds, including, but not limited to,saccharide-based, lipid-based, peptide-based, polypeptide-based andnucleic acid-based compounds. Synthetic compound libraries and librariesof natural compounds in the form of bacterial, fungal, plant, and animalextracts are commercially available from a number of sources. Inaddition, natural and synthetically libraries can be produced, ifdesired, according to routine methods, e.g., by standard extraction andfractionation methods. Furthermore, if desired, any library or compoundis readily modified using standard chemical, physical, or biochemicalmethods.

When a crude extract is found to have a desired activity, furtherfractionation of the positive lead extract may be necessary to isolatechemical constituents responsible for the observed effect. The goal ofthe extraction, fractionation, and purification process is the carefulcharacterization and identification of a chemical entity within thecrude extract having the desired activity. The disclosed assays can alsobe used to purify the active component and to test derivatives thereof.Methods of fractionation and purification of such heterogenous extractsare known in the art. If desired, compounds shown to be useful agentsfor treatment are chemically modified according to methods known in theart. Compounds identified as being of therapeutic value may besubsequently analyzed using appropriate in vitro or animal models.

Candidate agents encompass numerous chemical classes, but are most oftenorganic molecules, e.g., small organic compounds having a molecularweight of more than 100 and less than about 2,500 daltons. Candidateagents contain functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, for example, atleast two of the functional chemical groups. The candidate agents oftencontain cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 SAPK Signaling Upregulates XBP1

Materials and Methods

Cell Culture

MEF cells were from American Type Tissue Collection (ATCC). These cellswere grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with10% fetal bovine serum (FBS), 10 U/ml penicillin and 1 μg/mlstreptomycin at 37° C. and 5% CO₂. Fao cells were maintained in RPMI1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS),10 U/ml penicillin and 1 μg/ml streptomycin.

Recombinant XBP1s

Recombinant XBP1s was produced at GenScript. XBP1s CDS together with6×His-TF tag at N-terminal was cloned into pGSC1 plasmid. The sequencesof His-IF tag (6×His+TF tag+3C protease cleavage site) are as follow:

(SEQ ID NO: 1) MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMN QQASAGLEVL FQGP.

For producing recombinant XBP1s protein, 5 ng pGSC1 plasmid containingXBP1s and His-TF tag was transformed into ArcticExpress™ (DE3) RP hoststain E. coli. The total protein was extracted and purified byNi-affinity resin.

Adenovirus Production and Infection

Adenovirus expressing XBP1s T48A, XBP1s S61A, XBP1s T48A/S61A andMKK6Glu were produced with ViraPower Adenoviral Expression System(Invitrogen) according to manufacturer's instruction. Briefly, pAD-XBP1sT48A, pAD-XBP1s S61A, pAd-XBP1s T48A/S61A and pAd-MKK6Glu werelinearized by restriction endonuclease digestion with PacI andtransfected to 293A cells by Lipofectamine. The media was changed withfresh media every other day until the cytopathic effect was observed.When cytopathic effect reached to 80%, cells were collected bycentrifugation. The pellet was resuspended in PBS and subjected tofreezing and thawing cycles at −80° C. and 37° C. for 4 times. Thesupernatant containing the virus was prepared by centrifugation at 4,000rpm for 20 min at room temperature. Ad-XBP1s and Ad-Cre were generatedas described previously (Ozcan, L., et al. (2009) Cell metabolism9:35-51). For infection, cells were incubated with adenovirus in reducedvolume of medium containing 1% FBS and antibiotics. Cells were gentlyrocked every 15 min for 1 h to increase efficiency of infection, andthen fresh medium were added and cells were incubated for additional 15h or 23 h.

Total Protein Extraction from Cells

Cells were lysed in lysis buffer (25 mM Tris-HCl, pH 7.4; 10 mM NaF; 10mM Na₄P₂O₇; 2 mM Na₃VO₄; 1 mM EGTA; 1 mM EDTA; 1% NP-40; 10 μg/mlLeupeptin; 10 μg/ml Aprotinin; 1 mM PMSF and 20 nM Okadaic acid). After20 min-rotation at 4° C., cell lysates were centrifuged at 13,200 rpmfor 20 min at 4° C. Supernatants were collected and proteinconcentration was quantified by using Protein Assay Kit (Bio-Rad). Theconcentrations of protein were normalized with lysis buffer to haveequivalent amounts of protein and volume. Protein was denatured byboiling at 100° C. for 5 min in Laemmli buffer. The lysates were cooledto room temperature before loading for western blot analysis.

Western Blot Analysis

Western blot analysis was performed as previously described (Ozcan, L.,et al. (2009) Cell metabolism 9:35-51). Samples from cell lysates ortissue lysates were resolved by SDS-PAGE and then transferred topolyvinylidene fluoride (PVDF) membrane. After 1 h blocking at roomtemperature using 10% blocking reagent (Roche), membrane was incubatedovernight with primary antibody in Tris-buffered saline solution/Tween(TBST) containing 10% blocking reagent at 4° C. After the incubation,membrane was washed three times in TBST and incubated with secondaryantibody for 1 h at room temperature. After three-time washing in TBST,membrane was developed using a chemiluminescence assay system (Roche)and exposed to Kodak films. Relative protein levels were quantified byImage J program.

For stripping, membrane was vigorously shaken in stripping buffer (62.5mM Tris-HCl, pH 6.7; 2% SDS; 100 mM 2-mecaptomethanol) at 50° C. for 20min. After stripping, membrane was washed three times in TBST.

Statistical Analysis

Data are presented as means±standard error of the mean (SEM).Statistical significance was calculated by Student's t test or bymultifactor analysis of variance (ANOVA), with factors of time,treatment, and in some cases, genotype. Non-significant interactionterms involving time were taken as an indication that treatmentcontrasts could be pooled across time. When ANOVA indicated asignificant difference among the groups, the groups were compared usinga stricter criterion for statistical significance according to theBonferroni rule (corrected p value=pair-wise p value x number ofgroups). Significance was accepted at the level of p<0.05 (*), p<0.01(**), or p<0.001 (***).

Results

XBP1s is a central regulator of ER homeostasis. To investigate whetheractivation of TNK or p38 MAPK affects XBP activity, XBP was expressed inMEFs by infecting them with XBP1s-expressing adenovirus (Ad-XBP1s), orwith LacZ-expressing adenovirus (Ad-LacZ; control). Subsequently, thecells were treated for two hours with increasing doses of anisomycin, anagent that increases JNK and p38 MAPK activation. Anisomycin treatmentled to a robust increase in XBP1s protein levels, but only in cellsinfected with Ad-XBP1s. No upregulation of XBP proteins was noted inAd-LacZ-infected control cells treated with up to 25 ng/ml anisomycin,showing that anisomycin by itself does not create ER stress nor does itinduce XBP1 splicing. A time course experiment demonstrated thatexposure of the XBP1s-expressing cells to anisomycin (25 ng/ml) leads toupregulation of XBP1s protein levels within the first 30 minutes. Todetermine whether an alternative way of activating SAPKs would alsoincrease XBP1s protein levels, XBP1s-expressing cells were treated for aperiod of two hours with increasing doses of TNFα. As with theanisomycin, TNFα stimulation markedly increased XBP1s protein levels. Astudy of the time course of this effect revealed that TNFα (10 ng/ml)also upregulates XBP1s levels within 30 minutes, And finally,undertaking the same experiment in Fao cells (a rat hepatoma cell line)revealed that Ad-XBP1s-infected Fao cells also respond to anisomycin orTNFα as do the MEFs, by dramatically upregulating their XBP1s levels.

Example 2 SAPK Signaling Increases XBP1 mRNA Stability NuclearTranslocation

Materials and Methods

Real-Time Quantitative PCR

Total RNA was extracted from cells or animal tissues using Trizolreagent (Invitrogen) and transcribed into cDNA using cDNA synthesis kit(Bio-Rad). The gene expression analysis was performed with iQ5Multicolor Real-Time PCR Detection System (Bio-Rad) with SYBR GreenSupermix (Bio-Rad). The mRNA level was normalized to 18S as a housekeeping gene. The primer sequences used were:

18S rRNA forward: (SEQ ID NO: 2) 5′-AGT CCC TGC CCT TTG TAC ACA-3′;18S rRNA reverse: (SEQ ID NO: 3) 5′-CGT TCC GAG GGC CTC ACT-3′;XBP1s forward: (SEQ ID NO: 4) 5′-GGTCTGCTGAGTCCGCAGCAGG-3′;XBP1s reverse: (SEQ ID NO: 5) 5′-AGGCTTGGTGTATACATGG-3′.

Cytoplasmic and Nuclear Protein Extraction

Cytoplasmic and nuclear protein fractions were extracted from cells byusing nuclear protein extraction kit from Active Motif (Carlsbad,Calif.). Cells were maintained in 10 cm tissue culture dishes fornuclear/cytoplasmic extraction. After removal of the media, cells werewashed with ice-cold phosphate buffered saline (PBS) containingphosphatase inhibitors. Subsequently, 3 ml of ice-cold PBS withphosphatase inhibitors was added and then cells were scrapped out of thedish. Collected cells were separated from PBS by centrifugation for 5minutes at 500 rpm and resuspended with 500 μl of 1× hypotonic buffer.After 15-minute incubation in hypotonic buffer on ice, 25 μl of supplieddetergent was added and cells were vortexed 10 seconds. Cells werecentrifuged for 30 seconds at 14,000×g and supernatant (cytoplasmicfraction) was saved for further analysis. Remaining pellet, whichcontains nuclei, were resuspended with 50 μl of provided complete lysisbuffer with 1 mM dithiothreitol (DTT), vortexed 10 seconds, andincubated for 30 minutes on ice. After 30-second vortexing and 10-minutecentrifugation at 14,000×g, the supernatant was collected and analyzedas nuclear fraction.

For liver tissue, a kit from Thermo Scientific (Rockford, Ill.) was usedaccording to the manufacturer's instruction. Liver tissues were cut intosmall pieces, washed with PBS, and separated from PBS by centrifugationat 500×g for 5 minutes. Collected tissues were resuspended bycompany-supplied CER I buffer and homogenized with a Dounce homogenizer.Homogenized tissues were vortexed and incubated on ice for 10 minutes.Following CER II buffer addition, tissues were vortexed for 5 seconds,incubated for 1 minute on ice, vortexed again, and then centrifuged for5 minutes at maximum speed in a microcentrifuge. The supernatant(cytoplasmic fraction) was saved for later analysis. The pellets wereresuspended with supplied NER buffer and undergone a series of multiplevortexing (15 seconds) and incubation on ice (10 minutes) for a total of40 minutes. After 10-minute centrifugation, the supernatant (nuclearfraction) was collected and analyzed with immunoblotting.

Results

To determine whether upregulation of XBP1s levels is due to an increasein the protein stability, cells were infected with Ad-XBP1s,subsequently treated with vehicle or anisomycin (25 ng/ml) for twohours, then further treated with cycloheximide (10 μg/ml) to inhibit theinitiation of translation. XBP1s levels were determined before additionof cycloheximide, as well as 10, 20, 40, 60 and 90 minutes after addingit. To analyze the degradation rate of XBP1s in vehicle- and oranisomycin-treated cells correctly, blots from vehicle-treated cellswere exposed longer than anisomycin-treated cells to have the sameamount of XBP1s signal at 0 time points of both groups. No differencesin the degradation rate of XBP1s protein in the vehicle- oranisomycin-treated cells was observed (FIG. 2A). These results excludethe possibility that anisomycin increases XBP1s protein levels byincreasing its stability.

However, upon examining XBP1s mRNA levels in the vehicle-treated andanisomycin-treated XBP expressing cells, a dramatic increase in mRNA ofXBP1s was noted in the anisomycin-treated cells (FIG. 2B). To evaluatethe possibility that anisomycin treatment increases the stability of XBPmRNA, the XBP expressing cells were pretreated with anisomycin (25ng/ml) for one hour, and the degradation pattern of XBP mRNA wasexamined after adding actinomycin D (10 μg/ml—to inhibit transcription).Anisomycin extended the half-life of the mRNA from around 15 minutes to60 minutes (FIG. 2C). Thus, activation of SAPK signaling increases thehalf-life of XBP mRNA. Furthermore, nuclear levels of XBP inanisomycin-treated cells were 141.8 times higher than in vehicle-treatedcells, but cytoplasmic levels of the protein were only increased 28.8times (FIGS. 2D-2E). Under normal conditions, the fold increase in XBP1sshould be similar in the cytoplasmic and nuclear compartments. Thefive-fold discrepancy in these values for the anisomycin-treated cellssuggested that anisomycin increases the efficiency of nucleartranslocation of XBP1s. When taken together, the results suggested thatSAPK signaling increases the mRNA stability of XBP1s and leads to itsnuclear translocation with a higher efficiency.

Example 3 XBP1 Upregulation Independent of JNK Activation

Materials and Methods

Plasmids

MKK7-JNK1-expressing plasmids were obtained from. Addgene (Cambridge,Mass.).

Results

To unravel the molecular mechanisms underlying the above observations,several different approaches were taken to explore whether activation ofJNK, which is one of the most dominant signaling elements in SAPKsignaling, affects XBP levels and nuclear translocation. First,Ad-LacZ-infected cells and Ad-XBP1s-infected cells were treated with aspecific JNK inhibitor (JNK inhibitor VIII, 10 μM) for 30 minutes, thenexposed to anisomycin (25 ng/ml) for two hours. The JNK inhibitor waseffective in completely abolishing anisomycin-induced JNK activation(analyzed via c-Jun phosphorylation), but had no effect onanisomycin-mediated upregulation of XBP1s protein levels. As the secondapproach, JNK1/2^(−/−) were stimulated with anisomycin, which also ledto a significant increase in XBP1s protein that was significantly higherthan that in anisomycin-treated control cells. And, while no JNKactivity was detectable in the JNK1,2^(−/−) cells that were treated withanisomycin, a marked increase in p38 MAPK phosphorylation was noted. Itis known that MAPK kinase kinase (MKK) 4 and 7 are upstream kinasesresponsible for JNK activation (Tournier, C., et al. (2001) Genes Dev15:1419-1426; Brancho, D., et al. (2003) Genes Dev 17:1969-1978) andanisomycin or TNFα cannot activate JNK in MKK4 and 7 double knock outcells (MKK4,7^(−/−)) (Tournier, C., et al. (2001) Genes Dev15:1419-1426; Schaeffer, H. J. & Weber, M. J. (1999) Mol Cell Biol19:2435-2444). In order to determine whether XBP1s can be regulated byanisomycin treatment in MKK4,7^(−/−) cells, these cells, along withtheir controls, were treated with anisomycin after XBP1s expression. Asfor the JNK1,2^(−/−) cells, anisomycin stimulation dramaticallyincreased XBP1s levels in MKK4,7^(−/−) cells, to a level that wassignificantly higher than that in wt control cells. JNK activity was notdetectable in the MKK4,7^(−/−) cells, whereas p38 MAPK activation wassignificantly increased. Furthermore, the experiments were repeated inJNK1,2^(−/−) and MKK4,7^(−/−) cells with TNFα. As with anisomycin,treatment of JNK1,2^(−/−) and MKK4,7^(−/−) cells with TNFα also led to asignificant increase in XBP1s levels. Finally, an MKK7-JNK1 fusionprotein, previously shown to be a specific activator of JNK (Lei, K., etal. (2002) Mol Cell Biol 22:4929-4942), was used to investigate whetheractivation of JNK alone can have effect on XBP1s. MKK7-JNK1 expressionin the cells significantly increased JNK activation. However,co-expression of MKK7-JNK1 with XBP1s did not increase protein levels ofXBP1s, indicating that the process is independent of JNK activation.

Example 4 XBP1 Upregulation Mediated by p38 MAPK

Materials and Methods

Plasmids

MKK6Glu-expressing plasmids were obtained from Addgene (Cambridge,Mass.). For producing adenovirus expressing MKK6Glu, MKK6Glu in pcDNA3.1was subcloned into pENTR3C (Invitrogen) at the sites of Not I and Kpn I.Primer sequences are: 5′-TTAAG GGTACCGGCGCCATGTCTCAGTCGA AAGGCAA-3′(forward, SEQ ID NO:6); 5′.-TTAAG GCGGCCGCTTATCATTAGTCTCCAA GAATCAG-3′(reverse, SEQ ID NO:7). The resulting MKK6Glu in pENTR3C was furthersubcloned into pAD (Invitrogen) by using LR clonase (Invitrogen) forgenerating adenovirus.

Results

p38 MAPK is another critical SAPK signaling element that is activatedduring ER stress conditions, and in the absence of JNK activation,higher levels of p38 MAPK activation were noted. The possibility thatp38 MAPK activation mediates the effects of anisomycin and TNFα on XBP1swas therefore examined. Ad-XBP1s-infected cells were treated with aspecific p38 MAPK inhibitor (SB203580) (10 μM for 30 minutes), thenstimulated with anisomycin (25 ng/ml) for two hours. The dramaticincrease in the XBP1s levels was completely abolished by pretreatment ofthe cells with a p38 MAPK inhibitor. These findings suggest thatanisomycin-induced upregulation of XBP levels is mediated via the p38MAPK pathway.

MKK3 and 6 are required for p38 MAPK activation, and in fact, p38 MAPKcannot be activated in MKK3,6^(−/−) cells (Brancho, D., et al. (2003)Genes Dev 17:1969-1978). In light of this, it was reasoned thatanisomycin should not induce an increase in XBP levels in MKK3,6^(−/−)cells—and indeed, no increase in XBP levels was noted whenAd-XBP1s-infected MKK3,6^(−/−) cells were treated with anisomycin,relative to levels in anisomycin-stimulated Ad-LacZ-infected controlcells. And, as previously reported (Brancho, D., et al. (2003) Genes Dev17:1969-1978), p38 MAPK activation was dramatically reduced inMKK3,6^(−/−) cells. Ad-XBP1s-infected p38α^(−/−) cells also lacked theability to upregulate XBP1s levels in response to anisomycinstimulation.

A constitutively active form of MKK6 (MKK6Glu), one of the upstreamkinases of p38 MAPK, was previously generated by mutation of Ser207 andSer211 to glutamate. Expression of MKK6Glu specifically activates p38MAPK (Raingeaud, J., et al. (1996) Mol Cell Biol 16:1247-1255). MKK6Gluwas used to assess whether activation of p38 MAPK alone is sufficient toincrease XBP1s protein levels. Cells were first transfected with MKK6Gluand/or XBP at the presence or absence of p38 MAPK inhibitor (10 μM).Expression of MKK6Glu significantly increased XBP1s protein levels,similar to findings with anisomycin and TNFα. However, inhibition of p38MAPK activity blocked the effect of MKK6Glu on XBP1s.

Example 5

p38 MAPK Regulates XBP1s mRNA Stability

Materials and Methods

Protein Degradation and mRNA Stability Analysis

MEF cells were infected with Ad-XBP1s. After 24 h post-infection, cellswere treated with anisomycin at 25 ng/ml or vehicle (DMSO) for 2 h.Translation initiation inhibitor cycloheximide (10 μg/ml, Sigma) wasadded to the medium. Cells were flash frozen in liquid nitrogen atvarious time points. Protein levels were determined via immunoblotting.

For mRNA stability determination, MEF cells were infected with Ad-XBP1s.Sixteen hour after the infection, cells were treated with anisomycin (25ng/ml) or vehicle (DMSO) for 1 h. Actinomycin D (10 μg/ml, Sigma) wasadded to the medium. Cells were flash frozen in liquid nitrogen atvarious time points. mRNA levels were determined via Q-PCR.

Results

To investigate whether the increased stability of XBP mRNA was indeedmediated via the p38 MAPK pathway, cells were pretreated first with ap38 MAPK inhibitor (10 μM) for 30 minutes, then treated with anisomycin(25 ng/ml) for one hour, and finally incubated with actinomycin D (10μg/ml) to inhibit mRNA transcription. mRNA abundance of XBP1s wasanalyzed at indicated time points (FIG. 3A). Inhibition of p38 MAPKcompletely blocked the prolongation of the half-life of XBP mRNA inducedby anisomycin stimulation (FIG. 3A).

p38 MAPK signaling has also been implicated in the regulation ofstability of several different mRNAs (Clark, A., et al. (2009) FrontBiosci 14:847-871). One of the main molecules that links p38 MAPKactivation to regulation of mRNA stability is MAPK-activated proteinkinase 2 (MK2) (Clark, A., et al. (2009) Front Biosci 14:847-871). MK2was previously shown to regulate activation of several RNA-bindingproteins that are involved in stabilization of mRNA. These includetristetraprolin (TTP), human antigen R (HuR: ELAV family RNA bindingprotein) or ARE/poly(U)-binding degradation factor (AUF1). In additionp38 MAPK can also regulate RNA stability by directly phosphorylatingKH-domain splicing regulatory protein (KSRP) (Clark, A., et al. (2009)Front Biosci 14:847-871).

To investigate that the p38 MAPK-mediated increase in mRNA stability ofXBP1s is due to MK2 activation, Ad-XBP1s-infected control and MK2^(−/−)cells were stimulated with anisomycin. XBP1s protein levels weredramatically reduced in MK2^(−/−) cells, indicating that increased mRNAstability is primarily mediated via MK2 activation. The mRNA levels ofXBP were next examined in control and MK2^(−/−) cells that were infectedwith Ad-XBP1s and stimulated with anisomycin for one hour. Anisomycinincreased the mRNA levels of XBP in a dose dependent manner in controlcells (FIG. 3B), but the increase was significantly lower in theMK2^(−/−) cells (FIG. 3B), supporting a dominant role for MK2 in p38MAPK-mediated upregulation of XBP mRNA stability. Furthermore, ananalysis of degradation rate of XBP mRNA in control and MK2^(−/−) cellsthat were either treated with vehicle or anisomycin (25 ng/ml)documented that the enhanced stability of XBP1s mRNA after anisomycintreatment is completely lost in MK2^(−/−) cells (FIG. 3C). There were nodifferences in the degradation rate of XBP1s mRNA betweenvehicle-treated control and MK2^(−/−) and anisomycin-treated MK2^(−/−)cells (FIG. 3C). Nevertheless, a significant, but much smaller,upregulation of XBP mRNA levels was noted in the MK2^(−/−) cells thatwere treated with anisomycin (FIG. 3B) This small increase may be due toan increase in transcription of endogenous XBP1s. In fact, XBP1s mayhave positive impact on its own transcription.

Example 6 AUF1 and KSRP RNA-Binding Proteins not Involved in XBP1s mRNAStability

Materials and Methods

Gene Silencing Experiments with Lentiviral shRNAs

shRNAs in pLKO vector targeting AUF1 and KSRP were from Sigma-Aldrich.The target sequences for AUF1 are:

(shRNA #1, SEQ ID NO: 8) TGAATGGAAGTATGACGTT; (shRNA #2, SEQ ID NO: 9)AGTGGTTATGGGAAAGTAT; (shRNA #3, SEQ ID NO: 10) GAGAGTGTAGATAAGGTCA;(shRNA #4, SEQ ID NO: 11) CAATGTTGGTCTTAGTAAA.

The target sequences for KSRP are: CTGAGAAGATTGCTCACAT (shRNA #1, SEQ IDNO:12); TTGGGAAGAGTATTACAAA (shRNA #2, SEQ ID NO:13);AGCAGATTGACCATGCAAA (shRNA #3, SEQ ID NO:14); GCATCCAGTTCAAGCAAGAT(shRNA #4, SEQ ID NO:15). For generating lentiviral particles, pLP1,pLP2, VSVG and either pLKO empty vector or pLKO-containing AUF1 or KSPRshRNA insert were transfected into 293T packaging cells by usingLipofectamine (Invitrogen). The medium was changed 24 hpost-transfection and the viral supernatants were collected three dayspost-transfection, aliquoted and stored at −80° C. For enhancinginfection efficiency of lentivirus, MEF cells were pretreated withpolybrene (8 μg/ml, Sigma-Aldrich) overnight, and then cells wereinfected with lentivirus and medium was changed 24 h after theinfections. After 48 h post-infection, puromycin (2 μg/ml,Sigma-Aldrich) was added for selecting stably-infected cells and onlypuromycin-resistant cells were used in experiments. Cells infected withthe lentivirus containing empty vector pLKO used as controls.

Results

Tristetraprolin (TTP) is a member of a small family of RNA-bindingproteins that recognize AU-rich elements (AREs), and is one of thebest-known RNA-binding proteins that is the target of MK2. It has beenimplicated in regulating the stability of a variety of mRNAs (Clark, A.,et al. (2009) Front Biosci 14:847-871). No classical AREs wereidentified in the mRNA of XBP1s. In order to determine whether TTP mighthave a role in stabilizing XBP1s mRNA, Ad-XBP1s-infected TTP^(−/−) cellswere treated with anisomycin (25 ng/ml) for one hour, then analyzed formRNA degradation after inhibition of transcription with actinomycin D(10 μg/ml). Results show that anisomycin is capable of increasing XBP1smRNA stability even in TTP^(−/−) cells (FIG. 9A): stimulation withanisomycin led to the same levels of upregulation in XBP1s protein inboth wt control and TTP^(−/−) cells.

To investigate the role of AUF1 in stabilizing XBP1s mRNA, fourlentiviral shRNAs were obtained for AUF1 and MEFs were individuallytransduced with one of four AUF1 lentiviral shRNAs. snRNA #3 was themost efficient in suppressing AUF1 expression (by more than 90%) (FIG.9B). After establishing stable AUF1 knock down and control cell lines(pLKO), the cells were infected with Ad-XBP1s and subsequentlystimulated with anisomycin. Depletion of AUF1 does not affectanisomycin-induced upregulation of XBP is levels, indicating that AUF1is not the RNA binding protein that is involved in stabilization ofXBP1s mRNA.

The role of KSRP in regulating stability of XBP1s mRNA was nextinvestigated using the same approach as above for AUF1. shRNAs for KSRPwere used it to establish a stable KSRP knock-down cell line, as well asa control (pLKO) cell line (FIG. 9C). Stimulation of KSRP-knocked downcells with anisomycin for two hours revealed that upregulation of XBP1sprotein levels was similar between the pLKO controls and KSRP-knockeddown cells. Thus, KSRP also does not play a role in XBP1s mRNAstability. And finally, the role of HuR in this process wasinvestigated, but HuR was not detected in MEF cells.

Example 7 p38 MAPK Phosphorylation of XBP1s at Thr48 and Ser61 Criticalfor Nuclear Translocation of XBP1s

Materials and Methods

Mass Spectrometric Analysis by LC-MS/MS

MEFs were infected with flag-tagged XBP1s expressing adenovirus andtreated with anisomycin (25 ng/ml) for 2 h. Cells were washed withice-cold PBS and lysed with RIPA buffer (50 mM Tris-HCl, pH 7.5; 2 mMEGTA; 03% CHAPS; 100 mM NaF; 10 mM Na₄P₂O₇; 1 mM Na₃VO₄; 10 μg/mlLeupeptin; 10 μg/ml Aprotinin; 2 mM PMSF and 20 nM Okadaic acid). Afterovernight incubation with anti-flag antibody, Protein A sepharose beadswere added for additional 2 h at 4° C. Immunoprecipates were washedthree times with RIPA buffer containing 150 mM NaCl and boiled for 5 minin 2× Laemmli buffer for elution of immunoprecipitated XBP1s. Sampleswere resolved in SDS-PAGE and stained with Coomassie blue (Bio-Rad).Protein from Coomassie-stained gel bands was digested with trypsin andthe resulting peptide mixtures were subjected to microcapillary liquidchromatography tandem mass spectrometry (LC-MS/MS). MS/MS spectra wereassigned by searching them with the XBP is protein sequence using theSEQUEST algorithm.

Plasmids

Plasmids for amino acid substitution mutants for XBP1s (T48A, S61A andT48A/S61A) were generated by PCR-based mutagenesis using pcDNA3.1-XBP1sor pENTR-XBP1s as template with a kit from Stratagene (La Jolla,Calif.).

Primer sequences for T48A are: (forward, SEQ ID NO: 16)5′-GGGTCGGAGGCGAGCGGGGCACCGCAGGCTCGCAAGCGG-3′;  (reverse, SEQ ID NO: 17)5′-CCGCTTGCGAGCCTGCGGTGCCCCGCTCGCCTCCGACCC-3′.Primer sequences for S61A are: (forward, SEQ ID NO: 18)5′-CAGCGGCTCACGCACCTGGCCCCGGAGGAGAAAGCGC-3′; (reverse, SEQ ID NO: 19)5′-GCGCTTTCTCCTCCGGGGCCAGGTGCGTGAGCCGCTG-3′.

Mutations at specific positions were confirmed by sequencing. Theresulting mutated XBP in pcDNA3.1 (Invitrogen) were used for transienttransfection assays and mutated XBP in pENTR3C were further subclonedinto pAD for producing adenovirus.

In Vitro Kinase Assay

5 μg of recombinant XBP was incubated with activated recombinant p38αMAPK (Cell Signaling) in kinase assay buffer (25 mM Tris-HCl, pH 7.5; 5mM β-glycerophosphate; 2 mM dithiothreitol; 0.1 mM Na₃VO₄, 10 mM MgCl₂)containing 5 μCi of [γ-³²P]ATP (PerkinElmer) and 10 μM cold-ATP for 30min at 30° C. in the presence or absence of 10 μM SB203580 (Calbiochem).GST-ATF2 fusion protein (Cell Signaling) was used as a positive control.The reactions were terminated by addition of Laemmli buffer and sampleswere separated by SDS-PAGE and transferred to PVDF membranes. Totalprotein levels were detected by immunoblotting and incorporated ³²P(phosphorylation) was visualized by autoradiography.

Results

The dramatic increase in the efficiency of migration of XBP1s to thenucleus when p38 MAPK is activated was unexpected, and suggested thatp38 MAPK directly phosphorylates XBP1s and triggers this enhancedtranslocation. A flag-tagged-XBP1s was therefore expressed in cells,which were stimulated with anisomycin (25 ng/ml) for two hours. This wasfollowed by immunoprecipitation of XBP1s, resolving with SDS-PAGE, andvisualizing with Coomassie blue staining. MS/MS analysis (used todetermine the phosphorylation sites on XBP1s) demonstrated thatanisomycin stimulation increases phosphorylation of XBP1s at Ser61,which is a conserved phosphorylation site (LSPE) for p38 MAPK (FIG. 4).One other phosphorylation site (Thr48) on XBP1s is also a conserved p38MAPK phosphorylation site, although it was not detected with the MS/MSanalysis.

To convincingly demonstrate that Ser61 is indeed phosphorylated afteranisomycin stimulation or via activation of p38 MAPK, and also to testwhether Thr48 is also a phosphorylation site, a phospho-specificantibody was developed against phosphorylated XBP1s on Ser61 or onThr48. Stimulation of XBP1s-expressing cells with anisomycin (25 ng/ml)greatly increased phosphorylation of the Thr48 residue. To unravel therole of this phosphorylation site the Thr48 residue was mutated toalanine (T48A), which completely eliminated the anisomycin-inducedphosphorylation of XBP1s^(Thr48) but did not affect the upregulation inXBP1s protein levels. These results also demonstrate that thephospho-specific XBP1s^(Thr48) antibody does not recognize theunphosphorylated XBP1s. Despite the fact that XBP1s and mutantXBP1s-T48A are upregulated to similar levels after anisomycinstimulation, phosphorylation is only recognized in the wt XBP1s. Similarresults were obtained from XBP1s^(Ser61) phosphorylation. Anisomycinstimulation strongly induced phosphorylation of XBP1s at Ser61, butmutation of Ser61 did not alter the anisomycin-induced upregulation ofXBP protein levels. As with the p-XBP1s^(Thr48) antibody, these resultsalso establish the fact that the p-XBP1^(Ser61) antibody is specific forXBP1s that is phosphorylated on Ser61.

To determine whether p38 MAPK can directly phosphorylate XBP1s, andwhether anisomycin-induced phosphorylation of XBP at Thr48 and Ser61 isdirectly mediated via p38 MAPK, a His/TF-tagged-XBP1s was first clonedinto a pGS21a bacterial expression vector, and the XBP1s fusion proteinpurified in a E. coli strain (ArcticExpress™ (DE3) RP host strain).Following successful purification of the XBP protein, an in vitro kinaseassay was performed with an already activated recombinant p38 MAPKprotein. Incubation of XBP1s with activated recombinant p38 MAPKsignificantly increased phosphorylation of XBP1s, however, addition of ap38 MAPK inhibitor to the kinase assay buffer completely reversed theeffect of activated p38 MAPK on XBP1s. As a control, the p38 MAPK kinaseassay was also done with ATF2. Results demonstrated that activatedrecombinant p38 MAPK is fully functional and phosphorylates ATF2, andthat this phosphorylation is inhibited by p38 MAPK inhibitor. Inaddition, an in vitro kinase assay was aimed at understanding whetherThr48 and Ser61 are indeed directly phosphorylated by p38 MAPK. Directimmunoblotting with use of the phospho-specific antibodies forp-XBP1s^(Thr48) and p-XBP1s^(Ser61) demonstrated that p38 MAPK directlyphosphorylates XBP on Thr48 and Ser61 residues in the in vitro kinaseassay. To understand the role of these phosphorylations, and to excludea possible overlapping function in regulation of XBP activity andnuclear translocation, a double mutant XBP was created at Thr48/Ser61residues.

The contribution of each phosphorylation site was then investigated onthe nuclear translocation of XBP1s. WT, T48A, S61A and T48A/S61A mutantXBP were expressed in the cells, anisomycin (25 ng/ml) was added, andtotal levels of cytoplasmic and nuclear XBP1s proteins were determined.Mutation of Thr48, of Ser61, or of both sites together, did not affectthe anisomycin-stimulated upregulation of XBP1s protein levels. However,mutation of the Thr48 residue decreased XBP1s nuclear translocation byabout 49%, mutation of the Ser61 residue decreased it by about 79%,while mutation of both sites almost completely blocked nucleartranslocation of XBP1s. In the mean time, mutation of Thr48 and Ser61led to accumulation of XBP1s in the cytoplasm indicating that the doublemutant XBP1s, despite the dramatic increase in the protein levels cannot migrate to the nucleus. These findings indicate that Thr48 and Ser61phosphorylation is crucial for nuclear translocation of XBP1s.

Example 8 p38 MAPK Activation and Signaling Necessary for XBP1s NuclearTranslocation

Materials and Methods

Total Protein Extraction from Tissue

Tissues were homogenized with a bench-top homogenizer (Polytron, PT2100)in ice-cold tissue lysis buffer (25 mM Tris-HCl, pH 7.4; 100 mM NaF; 50mM Na₄P₂O₇; 10 mM Na₃VO₄; 10 mM EGTA; 10 mM EDTA; 1% NP-40; 10 μg/mlLeupeptin; 10 μg/ml Aprotinin; 2 mM PMSF and 20 nM Okadaic acid). Afterhomogenization, lysates were rotated for 1 h at 4° C. and then subjectedto centrifugation at 13,200 rpm for 20 min at 4° C. The lipid layer wasremoved and the supernatant was transferred into Eppendorf tubes forcentrifugation. This process was repeated for 2 times to get rid oflipid completely. Protein concentration was quantified by using ProteinAssay Kit (Bio-Rad). Equivalent protein concentration in each sample wasprepared and boiled at 100° C. for 5 min in Laemmli buffer. The lysateswere cooled to room temperature before loading for western blotanalysis.

XBP1 Splicing Assay

XBP1 splicing assay was performed by PCR with cDNA as template. The PCRconditions were as follows: 94° C. for 3 min; 29 cycles of 94° C. for 30sec, 58° C. for 30 sec, and 72° C. for 30 sec; and 72° C. for 3 min. Theprimer sequences are:

Forward: (SEQ ID NO: 20) 5′-ACACGCTTGGGAATGGACAC-3′; Reverse:(SEQ ID NO: 21) 5′-CCATGGGAAGATGTTCTGGG-3′.

Results

XBP1s is generated in the liver when an animal is refed after a fastingperiod (Park, S. W., et al. (2010) Nature Med 16:429-437), likely mainlydue to increased metabolic overloading. To create a metabolicoverloading model in cells, following a 16 h starvation in mediumwithout fetal bovine serum (FBS), cells were reincubated for 15, 30 and60 minutes with medium containing 10% FBS, following which total andnuclear proteins or mRNA were isolated and used to analyze variousparameters, including p38 MAPK activation, XBP1s nuclear migration, andsplicing. When the cells were starved, then stimulated with mediumcontaining 10% FBS, there was a robust increase in XBP1s splicing andnuclear translocation, as well as in activation of p38 MAPK (FIG. 10A).However, inhibition of p38 MAPK activity by pretreatment of the starvedcells with p38 MAPK inhibitor (10 μM) before addition of 10% FBScompletely eliminated XBP1s nuclear translocation without altering XBP1ssplicing, or without affecting the total amount of XBP is that isproduced in the cells (FIG. 10A). The same experiment was then repeatedin p38α^(−/−) and MKK3,6^(−/−) cells. FBS-induced XBP1s nucleartranslocation was dramatically reduced in p38α^(−/−) cells compared tocontrol cells, despite the fact that XBP1s splicing and protein levelswere similar in control and p38α^(−/−) cells (FIG. 10B). Moreover,PBS-induced XBP1s nuclear translocation was severely impaired in theMKK3,6^(−/−) cells (FIG. 10C). Together, these results indicate thatactivation of p38 MAPK is necessary for XBP1s nuclear translocation.

To determine whether re-feeding after a fasting period induces p38 MAPKactivation and XBP1s phosphorylation in the, wild-type (wt) lean micewere fasted for 24 hours and re-fed for one hour. p38 MAPKphosphorylation was significantly increased in the liver followingrefeeding, and XBP1s total proteins were upregulated. Also,phosphorylation of XBP1s at Thr48 and Ser61 was markedly increased afterre-feeding, indicating that XBP1s is phosphorylated at these sitesduring the re-feeding process and that refeeding leads to a robustupregulation in nuclear levels of XBP1s.

XBP1s cannot migrate to the nucleus in the liver of obese mice duringrefeeding (Park, S. W., et al. (2010) Nature Med 16:429-437).Consistently, re-feeding ob/ob mice after the fasting period does notincrease p38 MAPK phosphorylation. XBP1s levels in total lysates areincreased, but there is no detectable phosphorylation of XBP1s^(Thr48)or XBP1s^(Ser61), and no detectable XBP1s protein in the nucleus.

To examine whether inhibition of p38 MAPK signaling alone blocks XBP isnuclear translocation in the mouse, 8-week-old wt C57BL6 lean male micewere treated for three days either with vehicle or with the p38 MAPKinhibitor (SB203580) (2 mg/kg/day, intraperitoneally), after which themice were starved for 24 hours and refed for one hour. XBP1s nuclearlevels significantly increased after refeeding in vehicle-treatedcontrol group. However, XBP1s nuclear translocation in the liver ofSB203580-treated mice after re-feeding was completely absent. Analysisof ATF2, a p38 MAPK target, revealed a dramatic reduction inphosphorylation, indicating that SB203580 successfully blockedactivation of p38 MAPK. XBP1s mRNA levels in the liver of re-fed micethat were either treated with vehicle or with the inhibitor, weresignificantly increased both in the vehicle-treated and SB203580-treatedgroups (FIG. 5A). Further analysis of mRNA levels of GRP78, which is anXBP1s-target gene, revealed that GRP78 expression was significantlyincreased in the vehicle-treated, but not in the inhibitor-treated group(FIG. 5B). When taken together, these results demonstrate that p38 MAPKsignaling is critical for XBP1s nuclear translocation.

Example 9 p38 MAPK Activation Reduced ER Stress and Increases InsulinSensitivity

Materials and Methods

Blood Glucose and Plasma Insulin Measurements

Mice were fasted for 6 h, after which their blood was analyzed forglucose measurement with a glucose meter (Bayer, Mishawaka, Ind.). Forinsulin analysis, mice were fasted for 24 h and plasma insulin weremeasured with an Ultra Sensitive Mouse Insulin ELISA kit from CrystalChem (Downers Grove, Ill.).

Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)

For GTT analysis, mice were intraperitoneally injected with D-glucose(0.5 g/kg body weight for ob/ob, 2 g/kg body weight for XBP1^(flox/flox)mouse) after an overnight fasting. Tail vein blood was collected at 0,15, 30, 60, 90 and 120 min following glucose injection and blood glucosewas measured with a glucose meter from Bayer. For ITT analysis, micewere fasted for 6 h (from 8 am to 2 pm) and intraperitoneally injectedwith recombinant human insulin (2 IU/kg for ob/ob mice) from Eli Lilly(Indianapolis, Ind.). Blood was taken from tail vein at 0, 15, 30, 60,90 and 120 min after insulin injection and blood glucose was measured.

Real-Time Quantitative PCR

Total RNA was extracted and analyzed as described above. The primersequences used were:

18S rRNA forward:  (SEQ ID NO: 22) 5′-AGTCCCTGCCCTTTGTACACA-3′;18S rRNA reverse:  (SEQ ID NO: 23) 5′-CGTTCCGAGGGCCTCACT-3′;ERdj4 forward:  (SEQ ID NO: 24) 5′-CCCCAGTGTCAAACTGTACCAG-3′;ERdj4 reverse:  (SEQ ID NO: 25) 5′-AGCGTTTCCAATTTTCCATAAATT-3′;GRP78 forward:  (SEQ ID NO: 26) 5′-TCATCGGACGCACTTGGAA-3′;GRP78 reverse:  (SEQ ID NO: 27) 5′-CAACCACCTTGAATGGCAAGA-3′;G6Pase forward:  (SEQ ID NO: 28) 5′-CCGGTGTTTGAACGTCATCT-3′;G6Pase reverse:  (SEQ ID NO: 29) 5′-CAATGCCTGACAAGACTCCA-3′;PEPCK forward:  (SEQ ID NO: 30) 5′-ATCATCTITGGTGGCCGTAG-3′;PEPCK reverse:  (SEQ ID NO: 31) 5′-ATCTTGCCCTTGTGTTCTGC-3′; PGC1αforward:  (SEQ ID NO: 32) 5′-TGATGTGAATGACTTGGATACAGACA-3′; PGC1αreverse:  (SEQ ID NO: 33) 5′-CAATGCCTGACAAGACTCCA-3′;Glucokinase forward:  (SEQ ID NO: 34) 5′-GAAAAGATCATTGGCGGAAA-3′;Glucokinase reverse:  (SEQ ID NO: 35) 5′-CCCAGAGTGCTCAGGATGTTAAG-3′;

Blood Alanine Transaminase (ALT) and Aspartate Transaminase (AST)Measurements

The blood from mice before and after adenoviral injection was collectedand blood ALT and AST levels were measured with ALT Color Endpoint Assayand AST Color Endpoint Assay kits from Bioo Scientific (Austin, Tex.) asinstructed by provided manuals.

Results

In addition to comparing fasting and refeeding states, basal p38 MAPKactivity was also investigated in the liver, muscle and white adiposetissues of lean and obese mice. p38 MAPK phosphorylation wassignificantly reduced in the liver, muscle and adipose tissues of ob/oband high fat diet-fed obese mice when compared to their leancounterparts (FIGS. 11A-11C). Previous observations also indicated thatp38 MAPK phosphorylation was reduced in the liver tumors of the micethat were obese (Park, E. J., et al. (2010) Cell 140:197-208).

To determine whether reactivation of p38 MAPK in the liver of obese anddiabetic mice would increase XBP1s phosphorylation, reduce ER stress,increase glucose tolerance and reduce blood glucose levels, MKK6Glu wascloned into an adenoviral vector, and adenovirus particles (Ad-MKK6Glu)produced. Infection of cells with Ad-MKK6Glu led to a significantupregulation in MKK6Glu levels when compared with the Ad-LacZ-infectedcontrol cells. Next, eight-week-old ob/ob mice were injected though thetail vein with 8×10⁶ PFU/g of Ad-MKK6Glu, or of Ad-LacZ. Six-hourfasting blood glucose levels on post-injection day three weresignificantly reduced in the Ad-MKK6Glu-injected group compared with theAd-LacZ-injected control mice (FIG. 6A). A significant reduction wasnoted in the circulating insulin levels of the MKK6Glu overexpressingob/ob mice, indicating that insulin sensitivity is increased (FIG. 6B).Moreover, as documented by glucose tolerance test, the glucose disposalrate in the Ad-MKK6Glu-injected group was significantly enhanced whencompared with the Ad-LacZ-injected group (FIGS. 6C-6D). Performance ofinsulin tolerance test (ITT) at post-injection day three also revealedan increased insulin sensitivity in the Ad-MKK6Glu-injected group (FIG.6E).

Analysis of liver samples from Ad-LacZ and Ad-MKK6Glu-injected ob/obmice documented that p38 MAPK phosphorylation and downstream signalingwere significantly increased in the MKK6Glu expressing ob/ob livers.Furthermore, XBP1s total protein levels, nuclear protein levels, andThr48 and Ser61 phosphorylations were markedly increased when MKK6Gluwas expressed in the liver of ob/ob mice. mRNA levels of XBP1s, andexpression of XBP1s-target genes (GRP78 and Erdj4), were alsosignificantly elevated, indicating increased XBP1s mRNA stability andactivity (FIGS. 6F-6H). IRE1α phosphorylation levels, which areindicative of ER stress, was dramatically reduced in MKK6Glu-expressingob/ob livers, showing that ER stress is indeed reduced by activation ofp38 MAPK pathway.

To investigate whether insulin sensitivity is altered in the livers ofAd-MKK6Glu-injected ob/ob mice when compared with the controls, insulin(0.75 IU/kg) was infused though portal vein into the livers of theAd-LacZ-injected and Ad-MKK6Glu-injected ob/ob mice on post-injectionday seven, and the activation of IR, IRS1 and Akt analyzed. Nodifferences were seen between the two groups (saline versus insulininfusion) in terms of insulin-stimulated tyrosine phosphorylation of IRbut there were significant increases in IRS1 tyrosine and Akt^(Thr308)phosphorylations (FIGS. 6I-6K). Furthermore, expression levels of genes(such as GK, G6Pase, PEPCK and PGC1α), which are involved in the glucosehomeostasis, revealed a significant down regulation when MKK6Glu wasexpressed in the livers of the ob/ob mice (FIG. 6L).

The extent of MKK6Glu expression in other tissues such as muscle andwhite adipose tissue (WAT) was also determined after tail vein injectionof the adenoviruses. As shown in FIG. 12A, the only significant increasein the expression of MKK6Glu was observed in the liver. In addition LacZexpression in the liver by tail vein injection of Ad-LacZ did not affectthe blood glucose levels of ob/ob mice (FIG. 12B). Furthermore tail veininjection of either Ad-LacZ or Ad-MKK6Glu did not alter the bodyweightand also did not lead to increase in liver function tests such as ASTand ALT (FIGS. 12C-12D).

Example 10 MKK6Glu-Mediated Enhancement of Glucose Homeostasis is XBP1sDependent

Materials and Methods

Results

To determine the role of XBP1s in MKK6Glu-mediated enhancement ofglucose homeostasis MKK6Glu was overexpressed in the liver of 16-weekhigh fat diet (HFD)-fed XBP1^(flox/flox) mice, while XBP1 wassimultaneously depleted with use of an adenovirus that expresses Crerecombinase (Ad-Cre). The first group of mice were injected with Ad-LacZ(1.58×10⁸ pfu/g), second group with Ad-MKK6Glu (0.08×10⁸ pfu/g) plusAd-LacZ (1.5×10⁸ pfu/g) and the third group with Ad-MKK6Glu (0.08×10⁸pfu/g) and Ad-Cre (1.5×10⁸ pfu/g). in parallel with the results obtainedfrom the experiments on the ob/ob mice, injection of Ad-MKK6Glu into theXBP1^(flox/flox) mice led to a significant decrease in blood glucoselevels (FIG. 7A). However, injection of Ad-Cre together with Ad-MKK6Glucompletely eliminated the effect of MKK6Glu on blood glucose levels,indicating that XBP1s plays a major role in the improved glucosehomeostasis mediated by MKK6Glu expression in the liver (FIG. 7A). Next,a glucose tolerance test was performed on post injection day 15. Glucosedisposal from circulation was significantly enhanced in theMKK6Glu-expressing XBP1^(flox/flox) mice, while depletion of XBP1 in theMKK6Glu-expressing mice eliminated this effect (FIGS. 7B-7C).

p38 MAPK activation, MKK6 and XBP1s nuclear levels was also analyzed.p38 MAPK activation as well as MKK6 was significantly increased in allthe MKK6Glu expressing groups. XBP1s nuclear translocation wassignificantly increased in the MKK6Glu expressing group, but completelyeliminated in the MKK6Glu plus Cre recombinase-expressing group. Theseresults demonstate that XBP1s is successfully depleted by Crerecombinase expression in the liver of XBP1^(flox/flox) mice.Furthermore, analysis of IRE1^(Ser724) phosphorylation revealed asignificant down regulation in the MKK6Glu expressing group, and thisdownregulation was abolished in the MKK6Glu plus Cre recombinaseexpressing group (FIG. 7D). Next, the expression of chaperones wasdetermined. MKK6Glu expression led to a significant upregulation in theexpression of Erdj4 and GRP78 but this effect was diminished in theXBP1s-depleted group (FIG. 7E). Finally, analysis of G6Pase, PEPCK andPGC1α mRNA levels revealed that MKK6-mediated down regulation of thesegenes were lost in the XBP1s-depleted mice (FIG. 7F). There was a slightdecrease in GK levels but not at significant levels (FIG. 7F).

Example 11 XBP1s Phosphorylation Required for Nuclear Translocation andEffect on Glucose Tolerance

Overexpression of XBP in the liver of obese mice increases glucosetolerance and significantly reduce the blood glucose levels (Zhou, Y.,et al. (2011) Nature Med 17(3):356-65). Therefore, the T48A/S61A doublemutant XBP1s, when expressed in the liver of obese mice, should nottranslocate to the nucleus and should not have any effect on glucosehomeostasis. To confirm, eight-week-old male ob/ob mice were injectedeither with Ad-LacZ (3.6×10⁷ pfu/g) or Ad-XBP1s (3.6×10⁷ pfu/g) orAd-XBP1s T48A/S61A (3.6×10⁷ pfu/g). Analysis of blood glucose levels onpost-injection day three showed that blood glucose of Ad-XBP1s-injectedob/ob mice were at euglycemia levels (FIG. 8A). However, expression ofT48A/S61A mutant XBP had no effect on blood glucose levels (FIG. 8A).Performance of GTT on post-injection day five revealed that mutation ofThr48 and Ser61 diminished the ability of XBP1s to enhance glucosetolerance (FIGS. 8B-8C). In parallel with recent observations (Zhou, Y.,et al. (2011) Nature Med 17(3):356-65) medium levels of XBP1s expressionin the liver did not alter insulin tolerance (FIG. 8D).

Nuclear levels of XBP1s were significantly increased in the livers ofAd-XBP1s-injected group, but there were no detectable mutant XBP in thenuclear fractions of the livers of Ad-XBP1s T48A/S61A-injected group.Analysis of total protein and mRNA levels of XBP and XBP1s T48A/S61Ademonstrated that both XBP1s' were expressed at similar amounts in theliver (FIG. 8E). Furthermore, upregulation of the chaperones, such asErdj4 and GRP78, seen after XBP1s expression, were diminished in thelivers of ob/ob mice that were expressing the mutant XBP1s (FIG. 8F-8G).These results provide further evidence that phosphorylation of XBP1s onT48/S61 is imperative for its function in vivo.

1. A method of reducing blood glucose in a subject, comprisingadministering to the subject a pharmaceutical composition comprising aspecific activator of mitogen-activated protein kinase kinase 6 (MKK3),mitogen-activated protein kinase kinase 6 (MKK4), mitogen-activatedprotein kinase kinase 6 (MKK6), p38 mitogen-activated protein kinase(p38MAPK), mitogen-activated kinase-activated protein kinase 2 (MK2), ora combination thereof in an effective amount to reduce blood glucose. 2.The method of claim 1, wherein the specific activator of MKK3 promotesphosphorylation of MKK6 at residue Ser189.
 3. The method of claim 1,wherein the specific activator of MKK4 promotes phosphorylation of MKK6at residues Ser257, Thr261, or a combination thereof.
 4. The method ofclaim 1, wherein the specific activator of MKK6 promotes phosphorylationof MKK6 at residues Ser207, Thr211, or a combination thereof.
 5. Themethod of claim 1, wherein the specific activator of p38MAPK promotesphosphorylation of p38MAPK at residues Thr180, Tyr182, or a combinationthereof.
 6. The method of claim 1, wherein the specific activator of MK2promotes phosphorylation of MK2 at residue T334.
 7. The method of claim1, wherein the specific activator of is an allosteric activator.
 8. Amethod of reducing blood glucose in a subject, comprising administeringto the subject a pharmaceutical composition comprising a specificactivator of X-box binding protein 1 (XBP1) in an effective amount toreduce blood glucose in the subject, wherein the specific activatorincreases phosphorylation of XBP1 on Thr48 and Ser61.
 9. The method ofclaim 8, wherein the subject has type II diabetes.
 10. The method ofclaim 8, wherein the subject is obese.
 11. The method of claim 8,wherein the pharmaceutical composition is administered at a non-toxicdosage, wherein an effective amount of the specific activator ismaintained in the subject for a period of at least 5 days.
 12. Themethod of claim 11, wherein the pharmaceutical composition isadministered for a period of at least 10 days.
 13. The method of claim11, wherein the pharmaceutical composition is an extended releaseformulation.
 14. A method of identifying an agent for reducing bloodglucose in a subject, comprising (a) contacting a sample comprisingX-box binding protein 1 (XBP1) with a candidate agent, (b) detectingphosphorylation of XBP1, wherein an increase in XBP1 phosphorylationcompared to a control identifies a candidate agent for reducing bloodglucose in a subject.
 15. The method of claim 14, wherein an increase inXBP1 phosphorylation at residues Thr48, Ser61, or a combination thereofcompared to a control identifies a candidate agent for reducing bloodglucose in a subject.
 16. A method of identifying an agent for reducingblood glucose in a subject, comprising (a) contacting cells comprisingX-box binding protein 1 (XBP1) with a candidate agent, (b) detectingcellular localization of XBP1, wherein an increase in XBP1 nucleartranslocation compared to a control, identifies an agent for promotingglucose homeostasis in a subject.
 17. A method of identifying an agentfor reducing blood glucose in a subject, comprising (a) contacting asample comprising p38 mitogen-activated protein kinase (p38MAPK) with acandidate agent, (b) detecting phosphorylation of p38MAPK, wherein anincrease in p38MAPK phosphorylation compared to a control identifies anagent for reducing blood glucose in a subject.
 18. The method of claim17, wherein an increase in p38MAPK phosphorylation at residues Thr180,Tyr182, or a combination thereof compared to a control identifies acandidate agent for reducing blood glucose in a subject.
 19. A method ofreducing blood glucose in a subject, comprising administering to thesubject a pharmaceutical composition comprising an agent identified bythe method of claim 14 in an effective amount to reduce blood glucose.20. The method of claim 1, wherein the subject has type II diabetes. 21.The method of claim 1, wherein the subject is obese.
 22. The method ofclaim 1, wherein the pharmaceutical composition is administered at anon-toxic dosage, wherein an effective amount of the specific activatoris maintained in the subject for a period of at least 5 days.